Nucleic acid molecules encoding a novel ectoparasite saliva protein

ABSTRACT

The present invention is directed to a novel product and method for isolating ectoparasite saliva proteins, and a novel product and method for detecting and/or treating allergic dermatitis in an animal. The present invention includes a saliva protein collection apparatus capable of collecting ectoparasite saliva proteins substantially free of contaminating material. The present invention also relates to ectoparasite saliva proteins, nucleic acid molecules having sequences that encode such proteins, and antibodies raised against such proteins. The present invention also includes methods to obtain such proteins and to use such proteins to identify animals susceptible to or having allergic dermatitis. The present invention also includes therapeutic compositions comprising such proteins and their use to treat animals susceptible to or having allergic dermatitis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/271,344, filed Oct. 14, 2002, which is a continuation of applicationis a continuation of U.S. patent application Ser. No. 08/809,423, filedMay 1, 1997, entitled now abandoned, which is a national stageapplication (35 U.S.C. 371) of PCT/US95/13200, filed Oct. 6, 1995, whichclaims the benefit of priority from U.S. patent application Ser. No.08/487,608, filed Jun. 7, 1995, now abandoned, U.S. patent applicationSer. No. 08/487,001, filed Jun. 7, 1995, now U.S. Pat. No. 5,795,862,and U.S. patent application Ser. No. 08/319,590, filed Oct. 7, 1994, nowU.S. Pat. No. 5,646,115, all of which are incorporated herein by thisreference.

FIELD OF THE INVENTION

The present invention relates to a novel product and method forisolating ectoparasite saliva proteins, and a novel product and methodfor detecting and/or treating allergic dermatitis in an animal.

BACKGROUND OF THE INVENTION

Bites from ectoparasites, in particular fleas, can cause ahypersensitive response in animals. In particular, hypersensitiveresponses to fleabites is manifested in a disease called flea allergydermatitis (FAD). Hypersensitivity refers to a state of alteredreactivity in which an animal, having been previously exposed to acompound, exhibits an allergic response to the compound upon subsequentexposures. Hypersensitive responses include Type I, Type II, Type IIIand Type IV hypersensitivities. Type I hypersensitivity is described asIgE-mediated hypersensitivity in which an allergen induces cross-linkageof IgE bound to Fc receptors on the surface of mast cells. Thiscross-linkage results in the degranulation of the mast cells. Type IIhypersensitivity is described as antibody-mediated cytotoxichypersensitivity in which antibodies bind to cell surface allergensresulting in cell destruction via complement activation. Type IIIhypersensitivity is described as immune complex-mediatedhypersensitivity in which allergen-antibody complexes deposit in varioustissues and induce inflammatory responses. A delayed hypersensitivereaction includes Type IV hypersensitivity which is described as acell-mediated hypersensitivity in which T lymphocytes (i.e., T cells)release cytokines that activate macrophages or cytotoxic T cells whichmediate cellular destruction.

A Type I hypersensitive response usually occurs within about 2 to 30minutes following exposure to an allergenic compound, which is usually asoluble allergen. Type II and III responses can occur from about 2 to 8hours following exposure to an allergenic compound. Alternatively, in adelayed hypersensitivity response, the allergic response by an animal toan allergenic compound typically is manifested from about 24 to about 72hours after exposure to the compound. During the 24-hour delay,mononuclear cells infiltrate the area where the agent is located. Theinfiltrate can include lymphocytes, monocytes, macrophages andbasophils. Lymphokines (e.g., interferon-γ) are produced which activatemonocytes or macrophages to secrete enzymes (e.g., proteases) whichcause tissue damage.

Foreign compounds that induce symptoms of immediate and/or delayedhypersensitivity are herein referred to as allergens. The term“allergen” primarily refers to foreign compounds capable of causing anallergic response. The term can be used interchangeably with the term“antigen,” especially with respect to a foreign compound capable ofinducing symptoms of immediate and/or delayed hypersensitivity. Factorsthat influence an animal's susceptibility to an allergen can include agenetic component and/or environmental exposure to an allergen. Animalscan be de-sensitized to an allergen by repeated injections of theallergen to which an animal is hypersensitive.

FAD can have manifestations of both immediate and delayed-typehypersensitivity. Typically, an immediate hypersensitive response in ananimal susceptible to FAD includes wheal formation at the site of afleabite. Such wheals can develop into a papule with a crust,representative of delayed-type hypersensitivity. Hypersensitivereactions to fleabites can occur in genetically pre-disposed animals aswell as in animals sensitized by previous exposure to fleabites.

Effective treatment of FAD has been difficult if not impossible toachieve. FAD afflicts about 15% of cats and dogs in flea endemic areasand the frequency is increasing each year. In a geographical area,effective flea control requires treatment of all animals. One treatmentinvestigators have proposed includes desensitization of animals usingflea allergens. However, reliable, defined preparations of fleaallergens are needed for such treatments.

Until the discovery of the novel formulations of the present invention,flea allergens responsible for FAD had not been clearly defined. Wholeflea antigen preparations have been used to diagnose and desensitizeanimals with FAD (Benjamini et al., 1960, pp. 214-222, ExperimentalParasitology, Vol. 10; Keep et al., 1967, pp. 425-426, AustralianVeterinary Journal, Vol. 43; Kristensen et al., 1978, pp. 414-423, Nord.Vet-Med, Vol. 30; Van Winkle, 1981, pp. 343-354, J. Amer. Animal Hosp.Assoc., Vol. 17; Haliwell et al., 1987, pp. 203-213, VeterinaryImmunology and Immunopathology, Vol. 15; Greene et al., 1993, pp. 69-74,Parasite Immunology, Vol. 15); PCT Publication No. WO 93/18788 byOpdebeeck et al.; and Van Winkle, pp. 343-354, 1981, J. Am. Anim. Hosp.Assoc., vol. 32. Available commercial whole flea extracts, however, areunpredictable and, therefore, have limited usefulness.

Prior investigators have suggested that products contained in fleasaliva might be involved in FAD and have also suggested methods toisolate such products: Benjamini et al., 1963, pp. 143-154, ExperimentalParasitology, Vol. 13; Young et al., 1963, pp. 155-166, ExperimentalParasitology 13, Vol. 13; Michaeli et al., 1965, pp. 162-170, J.Immunol., Vol. 95; and Michaeli et al., 1996, pp. 402-406, J. Immunol.,Vol. 97. These investigators, however, have characterized the allergenicfactors of flea saliva as being haptens having molecular weights of lessthan 6 kilodaltons (kD). That they are not proteins is also supported bythe finding that they are not susceptible to degradation when exposed tostrong acids (e.g., 6 N hydrochloric acid) or heat. Some of theparticular low molecular weight allergenic factors have also beencharacterized as being a highly fluorescent aromatic fraction (Young etal., ibid.). In addition, studies by such investigators have indicatedthat in order to be allergenic, such factors need to be associated withadjuvants and/or carriers, such as collagen or portions of the membraneused to collect the oral secretions. Moreover, the methods described tocollect flea saliva factors were difficult and unpredictable.Furthermore the factors isolated by these methods were typicallycontaminated with material from the fleas, their culture medium or theskin-based membranes used to allow the fleas to feed.

Thus, there remains a need to more clearly define flea saliva allergenscapable of inducing a hypersensitive response in animals. In addition,there remains a need to develop a method to collect substantially pureflea saliva allergens which provide predictable and less expensivepreparations of allergens useful for desensitizing animals subject to,or having, FAD.

SUMMARY OF THE INVENTION

The present invention relates to, in one embodiment, a formulationcomprising at least one isolated ectoparasite saliva protein, in whichthe ectoparasite saliva protein comprises at least a portion of an aminoacid sequence, in which the portion is encoded by a nucleic acidmolecule capable of hybridizing under stringent conditions with anucleic acid molecule that encodes a flea saliva protein present in fleasaliva extract FS-1, FS-2 and/or FS-3. Preferred flea saliva proteinsinclude fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1,fspG2, fspG3, fspH, fspI, fspJ1, fspj2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and/or fspN3. In addition, the flea saliva proteinof the formulation can include at least a portion of an amino acidsequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:54 and/or SEQ ID NO:56.

Another embodiment of the present invention includes a formulationcomprising at least one isolated ectoparasite saliva protein, in whichthe ectoparasite saliva protein comprises at least a portion of an aminoacid sequence, in which the portion is encoded by a nucleic acidmolecule capable of hybridizing under stringent conditions with anucleic acid molecule that encodes a flea saliva protein represented asa protein peak in FIG. 2.

One aspect of the present invention includes a formulation comprising anectoparasite saliva product, in which the formulation, when submitted toTris glycine SDS-PAGE, comprises a fractionation profile as depicted ina FIG. 1B, lane 13 and/or FIG. 1B, lane 14.

Yet another embodiment of the present invention includes a formulationcomprising at least one isolated ectoparasite saliva productsubstantially free of contaminating material, the formulation beingproduced by a process comprising: (a) collecting ectoparasite salivaproducts on a collection means within a saliva collection apparatuscontaining ectoparasites, the apparatus comprising (i) a housingoperatively connected to a chamber, the chamber having an ambienttemperature warmer than the housing thereby forming a temperaturedifferential between the housing and the chamber, the housing beingcapable of retaining ectoparasites, and (ii) an interface between thehousing and the chamber, the interface comprising ((a)) a means capableof collecting at least a portion of saliva products deposited byectoparasites retained in the apparatus and ((b)) a barrier meanscapable of substantially preventing contaminating material fromcontacting the collection means, in which the temperature differentialattracts ectoparasites retained in the housing to attempt to feedthrough the barrier means and collection means and, thereby, depositsaliva products on the collection means; and (b) extracting the salivaproducts from the collection means to obtain the formulation. Alsoincluded in the present invention is such an apparatus and use such anapparatus to produce formulations comprising flea saliva productssubstantially free of contaminating material.

Another aspect of the present invention includes an isolated nucleicacid molecule capable of hybridizing under stringent conditions with agene encoding a flea saliva protein present in flea saliva extract FS-1,FS-2 and/or FS-3, including, but not limited to fspA, fspB, fspC1,fspC2, fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI, fspJ1,fspj2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and/or fspN3. Inparticular, the nucleic acid molecule is capable of hybridizing understringent conditions with nucleic acid sequence SEQ ID NO:20, SEQ IDNO:24, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:52 or SEQ IDNO:55. Also included in the present invention are recombinant moleculesand recombinant cells having a nucleic acid molecule of the presentinvention.

Also included in the present invention is a method for producing atleast one ectoparasite saliva protein, comprising: (a) culturing a celltransformed with at least one nucleic acid capable of hybridizing understringent conditions with a gene encoding a flea saliva protein presentin flea saliva extract FS-1, FS-2, and/or FS-3 to produce the protein;and (b) recovering the ectoparasite saliva proteins.

Another aspect of the present invention includes an antibody capable ofselectively binding to an ectoparasite saliva product, or mimetopethereof.

Yet another aspect of the present invention includes a therapeuticcomposition for treating allergic dermatitis comprising any of theformulations disclosed herein. In particular, the therapeuticcomposition is useful for treating flea allergy dermatitis, mosquitoallergy dermatitis and/or Culicoides allergy dermatitis. Moreover,particular flea saliva proteins to include in a therapeutic compositioninclude at least a portion of at least one of the following flea salivaproteins: fspE, fspf, fspG1 fspG2 fspG3, fspH, fspI, fspJ1, fspj2, fspK,fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and/or fspN3. The presentinvention also includes a method to desensitize a host animal toallergic dermatitis, comprising administering to the animal atherapeutic composition.

The present invention further relates to an assay kit for testing if ananimal is susceptible to or has allergic dermatitis, the kit comprising:(a) a formulation as disclosed herein; and (b) a means for determiningif the animal is susceptible to or has allergic dermatitis, in which themeans comprises use of the formulation to identify animals susceptibleto or having allergic dermatitis.

According to the present invention, a method can be used to identify ananimal susceptible to or having allergic dermatitis, the methodcomprising: (a) administering to a site on the animal a formulation ofthe present invention and administering to a different site on theanimal a control solution selected from the group consisting of positivecontrol solutions and negative control solutions; and (b) comparing areaction resulting from administration of the formulation with areaction resulting from administration of the control solution. Theanimal is determined to be susceptible to or to have allergic dermatitisif the reaction to the formulation is at least as large as the reactionto the positive control solution. The animal is determined not to besusceptible to or not to have allergic dermatitis if the reaction to theformulation is about the same size as the reaction to the negativecontrol solution. In particular, the method can detect immediatehypersensitivity and/or delayed hypersensitivity.

Also according to the present invention, a method can be used toidentify an animal susceptible to or having allergic dermatitis bymeasuring the presence of antibodies indicative of allergic dermatitisin the animal, the method comprising: (a) contacting a formulation ofthe present invention with a body fluid from the animal under conditionssufficient for formation of an immunocomplex between the formulation andthe antibodies, if present, in the body fluid; and (b) determining theamount of immunocomplex formed, in which formation of the immunocomplexindicates that the animal is susceptible to or has allergic dermatitis.In particular, the method can be used to detect IgE antibodies as anindicator of immediate hypersensitivity in the animal.

The present invention also includes a method for prescribing treatmentfor allergic dermatitis, comprising: (a) identifying an animal that issusceptible to or has allergic dermatitis by an in vivo or in vitroassay comprising a formulation of the present invention; and (b)prescribing a treatment comprising administering formulation of thepresent invention to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the resolution of flea saliva proteins by reducing16% Tris glycine SDS-PAGE.

FIG. 1B illustrates the resolution of flea saliva proteins, FS-1 andFS-2 by reducing 16% Tris glycine SDS-PAGE.

FIG. 1C illustrates the resolution of fspN by reducing 16% Tris glycineSDS-PAGE.

FIG. 2 illustrates the resolution of flea saliva proteins using highpressure liquid chromatography.

FIG. 3 illustrates the peaks obtained from reverse phase HPLC resolutionof proteolytic fragments of fspH protein digested with EndoproteinaseAsp-N.

FIG. 4A illustrates a cross-section of a flea saliva collectionapparatus of the present invention.

FIG. 4B illustrates a blow-out of a flea saliva collection apparatus ofthe present invention.

FIG. 5 illustrates the relative size of wheals produced 15 minutes afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

FIG. 6 illustrates the relative induration of wheals 6 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

FIG. 7 illustrates the relative erythema of wheals 6 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

FIG. 8 illustrates the relative induration of wheals 24 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

FIG. 9 illustrates the relative erythema of wheals 24 hours afterinjection of various flea saliva protein formulations intoflea-sensitized dogs.

FIG. 10 depicts ELISA results measuring anti-flea saliva IgE antibodiesin the sera of flea sensitized dogs.

FIGS. 11A and 11B depict ELISA results measuring anti-flea saliva IgEantibodies in the serum of a flea sensitized dog and the lack thereof inheartworm infected dogs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a novel product and method for diagnosingand treating allergic dermatitis of animals to ectoparasites. Theinvention is particularly advantageous in that it provides for a uniqueformulation of ectoparasite saliva products sufficiently free ofcontaminants such as blood proteins, fecal material and larval culturemedium, to be useful in diagnosis and therapy of allergies caused byectoparasites. In addition, the present invention includes ectoparasitesaliva products having other activities, important, for example, in aflea's ability to feed and/or counteract a host's resistance to fleas,such as products having clotting, anti-coagulant, protease,phospholipase, prostaglandin, anti-complement, other immunosuppressant,phosphatase, apyrase, vasoactive, and/or anti-inflammatory activities.Included among flea saliva products are products, such as, but notlimited to, proteases, that are regurgitated by the flea that originatedin other organs, such as, but not limited to, the midgut.

The invention is also particularly advantageous in that it provides anapparatus and method for reproducibly and efficiently isolatingectoparasite saliva products substantially free of contaminatingmaterial.

According to the present invention, ectoparasites are external livingparasites that attach and feed through the skin of a host animal.Ectoparasites include parasites that live on a host animal and parasitesthat attach temporarily to an animal in order to feed. Also, accordingto the present invention, ectoparasite saliva refers to the materialreleased from the mouth of an ectoparasite when the ectoparasiteattempts to feed in response to a temperature differential, such asexists in an apparatus of the present invention. Ectoparasite salivaincludes ectoparasite saliva products. Ectoparasite saliva products ofthe present invention comprise the portion of ectoparasite saliva boundto a collecting means of the present invention (described in detailbelow), herein referred to as ectoparasite saliva components. As such,ectoparasite saliva products also include the portion of ectoparasitesaliva extracted from a collecting means of the present invention,herein referred to as ectoparasite saliva extract. Included inectoparasite saliva extracts are ectoparasite saliva proteins which canbe isolated using, for example, any method described herein.Ectoparasite saliva extracts of the present invention can also includeother ectoparasite saliva products, such as, prostaglandins and otherpharmacologically active molecules.

One embodiment of the present invention is a formulation that containsectoparasite saliva products that can be used to diagnose and/or treatanimals susceptible to or having (i.e., suffering from) allergicdermatitis. Preferred types of allergic dermatitis to diagnose and/ortreat using ectoparasite saliva products of the present inventioninclude flea allergy dermatitis, Culicoides allergy dermatitis andmosquito allergy dermatitis. A preferred type of allergic dermatitis todiagnose and/or treat using ectoparasite saliva products of the presentinvention is flea allergy dermatitis. As used herein, an animal that issusceptible to allergic dermatitis refers to an animal that isgenetically pre-disposed to developing allergic dermatitis and/or to ananimal that has been primed with an antigen in such a manner thatre-exposure to the antigen results in symptoms of allergy that can beperceived by, for example, observing the animal or measuring antibodyproduction by the animal to the antigen. As such, animals susceptible toallergic dermatitis can include animals having sub-clinical allergicdermatitis. Sub-clinical allergic dermatitis refers to a condition inwhich allergy symptoms cannot be detected by simply observing an animal(i.e., manifestation of the disease can include the presence ofanti-ectoparasite saliva protein antibodies within an affected animalbut no dermatitis). For example, sub-clinical allergic dermatitis can bedetected using in vivo or in vitro assays of the present invention, asdescribed in detail below. Reference to animals having allergicdermatitis includes animals that do display allergy symptoms that can bedetected by simply observing an animal and/or by using in vivo or invitro assays of the present invention, as described in detail below.

One embodiment of the present invention is a formulation that includesone or more isolated ectoparasite saliva proteins. According to thepresent invention, an isolated protein is a protein that has beenremoved from its natural milieu. An isolated ectoparasite saliva proteincan, for example, be obtained from its natural source, be produced usingrecombinant DNA technology, or be synthesized chemically. As usedherein, an isolated ectoparasite saliva protein can be a full-lengthectoparasite saliva protein or any homologue of such a protein, such asan ectoparasite saliva protein in which amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., by glycosylation,phosphorylation, acetylation, myristylation, prenylation, palmitation,amidation and/or addition of glycosylphosphatidyl inositol). A homologueof an ectoparasite saliva protein is a protein having an amino acidsequence that is sufficiently similar to a natural ectoparasite salivaprotein amino acid sequence that a nucleic acid sequence encoding thehomologue is capable of hybridizing under stringent conditions to (i.e.,with) a nucleic acid sequence encoding the natural ectoparasite salivaprotein amino acid sequence. As used herein, stringent hybridizationconditions refer to standard hybridization conditions under whichnucleic acid molecules, including oligonucleotides, are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. The minimal size of a proteinhomologue of the present invention is a size sufficient to be encoded bya nucleic acid molecule capable of forming a stable hybrid with thecomplementary sequence of a nucleic acid molecule encoding thecorresponding natural protein. As such, the size of the nucleic acidmolecule encoding such a protein homologue is dependent on nucleic acidcomposition and percent homology between the nucleic acid molecule andcomplementary sequence as well as upon hybridization conditions per se(e.g., temperature, salt concentration, and formamide concentration).The minimal size of such nucleic acid molecules is typically at leastabout 12 to about 15 nucleotides in length if the nucleic acid moleculesare GC-rich and at least about 15 to about 17 bases in length if theyare AT-rich. As such, the minimal size of a nucleic acid molecule usedto encode an ectoparasite saliva protein homologue of the presentinvention is from about 12 to about 18 nucleotides in length. There isno limit, other than a practical limit, on the maximal size of such anucleic acid molecule in that the nucleic acid molecule can include aportion of a gene, an entire gene, or multiple genes, or portionsthereof. Similarly, the minimal size of an ectoparasite saliva proteinhomologue of the present invention is from about 4 to about 6 aminoacids in length, with preferred sizes depending on whether afull-length, multivalent (i.e., fusion protein having more than onedomain each of which has a function), or functional portions of suchproteins are desired.

Ectoparasite saliva protein homologues can be the result of allelicvariation of a natural gene encoding an ectoparasite saliva protein. Anatural gene refers to the form of the gene found most often in nature.Ectoparasite saliva protein homologues can be produced using techniquesknown in the art including, but not limited to, direct modifications toa gene encoding a protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

Preferred ectoparasite saliva proteins of the present invention,including homologues thereof, are capable of detecting and/or treatingallergic dermatitis resulting from the bites of ectoparasites. Apreferred ectoparasite saliva protein homologue includes at least oneepitope capable of eliciting a hypersensitive response to the naturalectoparasite saliva protein counterpart. An ectoparasite saliva proteinhomologue can also include an epitope capable of hyposensitizing ananimal to the natural form of the protein. The ability of anectoparasite saliva protein homologue to detect and/or treat (i.e.,immunomodulate or regulate by, for example, desensitizing) thehypersensitivity of an animal susceptible to or having allergicdermatitis, can be tested using techniques known to those skilled in theart. Such techniques include skin tests and immunoabsorbent assays asdescribed in detail below. Additional preferred ectoparasite salivaproteins of the present invention have other activities that includeactivities important for feeding and survival of the ectoparasite.

In one embodiment, a formulation of the present invention can comprise aprotein having at least a portion of an isolated ectoparasite salivaprotein. According to the present invention, “at least a portion of anectoparasite saliva protein” refers to a portion of an ectoparasitesaliva protein encoded by a nucleic acid molecule that is capable ofhybridizing, under stringent conditions, with a nucleic acid encoding afull-length ectoparasite saliva protein of the present invention.Preferred portions of ectoparasite saliva proteins are useful fordetecting and/or treating allergic dermatitis resulting from the bitesof ectoparasites. Additional preferred portions have activitiesimportant for flea feeding and survival. Suitable sizes for portions ofan ectoparasite saliva protein of the present invention are as disclosedfor saliva protein homologues of the present invention.

As will be apparent to one of skill in the art, the present invention isintended to apply to all ectoparasites. A formulation of the presentinvention can include saliva products from any ectoparasites. Apreferred ectoparasite of the present invention from which to isolatesaliva products (including proteins), and/or from which to identifyproteins that can then be produced recombinantly or synthetically,include arachnids, insects and leeches. More preferred ectoparasitesfrom which to obtain saliva products include fleas; ticks, includingboth hard ticks of the family Ixodidae (e.g., Ixodes and Amblyomma) andsoft ticks of the family Argasidae (e.g., Ornithodoros, such as O.parkeri and O. turicata); flies, such as midges (e.g., Culicoides),mosquitos, sand flies, black flies, horse flies, horn flies, deer flies,tsetse flies, stable flies, myiasis-causing flies and biting gnats;ants; spiders, lice; mites; and true bugs, such as bed bugs and kissingbugs, including those carrying Chagas disease. Even more preferredectoparasite saliva products include those from fleas, mosquitos,midges, sandflies, blackflies, ticks and Rhodnius, with products fromfleas, mosquitos and Culicoides being even more preferred.

A particularly preferred formulation of the present invention includesflea saliva proteins. Preferred flea saliva products include those fromCtenocephalides, Xenopsylla, Pulex, Tunga, Nosopsyllus, Diamanus,Ctopsyllus and Echidnophaga fleas, with saliva products fromCtenocephalides canis and Ctenocephalides felis fleas being even morepreferred. For the purposes of illustration, many of the followingembodiments discuss flea saliva proteins. Such discussion of flea salivaproteins is not intended, in any way, to limit the scope of the presentinvention.

In one embodiment, a formulation of the present invention issubstantially free from contaminating material. Contaminating materialcan include, for example, ectoparasite fecal material, blood proteinsfrom previous meals taken by an ectoparasite (e.g., fetuin, ferritin,albumin, hemoglobin and other large blood proteins), ectoparasitecuticular debris, and ectoparasite larval culture medium (e.g., blood,mouse food and sand). As used herein, a formulation that issubstantially free of contaminants is a formulation that without furtherpurification can be used as a diagnostic or therapeutic agent withoutcausing undesired side effects. Preferably, a formulation substantiallyfree from contaminating material comprises less than about 50 percentcontaminating material, more preferably less than about 10 percentcontaminating material, and even more preferably less than about 5percent contaminating material. As such, a formulation of the presentinvention preferably comprises at least about 50 percent flea salivaproducts, more preferably at least about 90 percent flea salivaproducts, and even more preferably at least about 95 percent flea salivaproducts. A formulation of the present invention substantially free ofcontaminating material can include a formulation not having any bloodcontaminants or flea midgut contents. A formulation substantially freeof contaminating material can be obtained using a saliva collectionapparatus of the present invention as described in detail below.

A formulation that is substantially free of contaminating material canbe identified by typical methods known to those of skill in the art. Forexample, the presence of contaminants can be identified by: (1)overloading and resolving a formulation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE); (2) resolving aformulation by a variety of chromatography techniques; (3) screening aformulation with antibodies capable of binding to specific contaminantsusing, for example, immunoblot or enzyme-linked immunoassay techniques;(4) resolving a formulation by capillary electrophoresis; or (5)screening a formulation using an assay to detect hemoglobin.

One embodiment of a formulation of the present invention includes atleast one or more flea saliva proteins having molecular weights rangingfrom about 6 kD to about 65 kD as determined by Tris-glycine SDS-PAGE,preferably using a 14% polyacrylamide gel and resolved using methodsstandard in the art. A preferred formulation includes one or more fleasaliva proteins having molecular weights ranging from about 6 kD toabout 55 kD. A more preferred formulation includes one or more proteinshaving elution (or migration) patterns as shown in FIG. 1.

In another embodiment, a formulation of the present invention includesat least one or more flea saliva proteins having molecular weightsranging from about 40 kD to about 300 kD as determined by Tris-glycineSDS-PAGE and resolved using methods standard in the art. Greater than50% of the flea saliva proteins contained in such a formulation have amolecular weights ranging from about 40 kD to about 55 kD, and appear tobe similar to fspN. A more preferred formulation includes one or moreproteins having elution (or migration) patterns as shown in FIG. 1.

In another embodiment, a formulation of the present invention includesone or more flea saliva proteins having basic isoelectric points, or pIvalues. An isoelectric pH, or pI, value refers to the pH value at whicha molecule has no net electric charge and fails to move in an electricfield. A preferred formulation of the present invention includesproteins having a pI value of at least about pI 8.5, and more preferablyof at least about pI 9.0. Flea saliva protein fspH, for example, has pIvalues ranging from about pI 8.5 to about pI 9.6, which may representheterogeneity in the proteins due to allelic variation in the fleapopulation from which the flea saliva proteins were collected.

In yet another embodiment, a formulation of the present inventionincludes at least a portion of one or more flea saliva products elutedfrom a collection means of the present invention. Examples of suchformulations include flea extracts FS-1, FS-2, and FS-3. The FS-1, FS-2and FS-3 fleas saliva extracts are produced according to the methoddescribed in detail in Example 2. According to the present invention,the terms FS-1 flea saliva extract, FS-2 flea saliva extract or FS-3flea saliva extract can be used interchangeably with the terms FS-1 fleasaliva product mixture, FS-2 flea saliva product mixture or FS-3 fleasaliva product mixture, respectively.

An FS-1 flea saliva extract includes a mixture of proteins (a) that,when submitted to reducing 16% Tris glycine SDS-PAGE, migrate as bandsas are shown in FIG. 1B, lane 13; and (b) that, when submitted toreverse phase high pressure liquid chromatography (HPLC), migrate aspeaks as are shown in FIG. 2. The peaks in FIG. 2 are obtained when theproteins included in FS-1 are collected using a saliva collectionapparatus of the present invention as described in detail below, andfurther resolved into protein peaks by passing the collected proteinsover a C4 HPLC column using 5% to 63% acetonitrile or 5.6% to 70%Solvent B at a flow rate of 0.8 milliliters per minute, in which SolventA is about 0.1% TFA in water and Solvent B is about 0.085% TFA in 90%acetonitrile. Referring to FIG. 2, the peaks are referred to anddepicted as peak A, peak B, peak C, peak D, peak E, peak F, peak G, peakH, peak I, peak J, peak K, peak L, peak M and peak N. Flea salivaproteins (or protein fragments) contained within such peaks are referredto as fspA, fspB, fspC, fspD, fspE, fspF, fspG, fspH, fspI, fspj, fspK,fspL, fspM and fspN. The peaks refer to the regions marked in FIG. 2 andit is to be noted that a peak does not necessarily contain just oneprotein (or protein fragment). Further resolution of proteins containedwithin the above-referenced peaks by, for example, amino acid sequencingor SDS-PAGE gel electrophoresis, has indicated that fspC includes atleast two proteins referred to as fspC1 and fspC2, fspD includes atleast two proteins referred to as fspD1 and fspD2, fspG includes atleast three proteins referred to as fspG1, fspG2 and fspG3, fspJincludes at least two proteins referred to as fspJ1 and fspj2, fspLincludes at least two proteins referred to as fspL1 and fspL2, fspMincludes at least two proteins referred to as fspM1 and fspM2, and fspNincludes at least three proteins and/or protein fragments referred to asfspN1, fspN2 and fspN3. At least partial amino acid sequences have beenobtained for a number of the flea saliva proteins as represented by SEQID NO:1 (a partial N- (amino-) terminal amino acid sequence of fspA),SEQ ID NO:2 (an amino acid sequence, beginning at the N-terminus, thatrepresents most of the fspH protein), SEQ ID NO:3 (a partial N-terminalamino acid sequence of an Endoproteinase Asp-N fragment of fspH, denotedfspHe), SEQ ID NO:4 (a partial N-terminal amino acid sequence of anEndoproteinase Asp-N fragment of fspH, denoted fspHh), SEQ ID NO:5 (apartial N-terminal amino acid sequence of an Endoproteinase Asp-Nfragment of fspH, denoted fspHj, which also represents a partialN-terminal amino acid sequence of fspH), SEQ ID NO:6 (a partialN-terminal amino acid sequence of fspI), SEQ ID NO:7 (a partialN-terminal amino acid sequence of fspJ1), SEQ ID NO:8 (a partialN-terminal amino acid sequence of fspJ2), SEQ ID NO:9 (a partialN-terminal amino acid sequence of fspL1), SEQ ID NO:10 (a partialN-terminal amino acid sequence of fspL2), SEQ ID NO:11 (a partialN-terminal amino acid sequence of fspN1), SEQ ID NO:12 (a partialN-terminal amino acid sequence of fspN2), SEQ ID NO:13 (a partialN-terminal amino acid sequence of fspN3), SEQ ID NO:14 (a partialN-terminal amino acid sequence of fspH), SEQ ID NO:25 (a translation ofthe nucleic acid sequence represented by SEQ ID NO:24, corresponding tofspI), SEQ ID NO:26 (an apparent full-length translation product offspI), SEQ ID NO:27 (a partial N-terminal amino acid sequence of fspB),SEQ ID NO:28 (a partial N-terminal amino acid sequence of fspG1), SEQ IDNO:29 (a partial N-terminal amino acid sequence of fspG2), SEQ ID NO:30(a partial N-terminal amino acid sequence of fspG3), SEQ ID NO:31 (apartial N-terminal amino acid sequence of an Endoproteinase Asp-Nfragment of fspN, denoted fspN(100-101)), SEQ ID NO:33 (a translationproduct, named PfspH₈₀, of the partial nucleic acid sequencecorresponding to fspH, named nfspH₂₄₂, denoted SEQ ID NO:32), SEQ IDNO:35 (a translation product, named PfspI₁₅₅ of the partial nucleic acidsequence corresponding to fspI, named nfspI₅₉₁, denoted SEQ ID NO:34),SEQ ID NO:51 (a translation product, named PfspN(A)₁₇₂, of the partialnucleic acid sequence of a fspN protein, named nfspN(A)₆₄₆, denoted SEQID NO:50), SEQ ID NO:53 (a translation product, named PfspN(B)153, ofthe partial nucleic acid sequence of a fspN protein named nfspN(B)₆₁₂,denoted SEQ ID NO:52), SEQ ID NO:54 (a partial apparent N-terminal aminoacid sequence, named PfspN(A)₅₆, of a fspN protein named PfspN(A)), andSEQ ID NO:56 (an apparent full-length translation product, namedPfspN(A)₃₉₈, of the apparent complete nucleic acid sequence of fspN3,named nfspN(A)₁₁₉₇, denoted SEQ ID NO:55). The details of how eachprotein was characterized is described in Examples 2 and 3.

An FS-2 flea saliva extract includes a mixture of proteins that, whensubmitted to reducing 16% Tris glycine SDS-PAGE, migrate as bands as areshown in FIG. 1B, lanes 14 and 15.

It is within the scope of the present invention that additional fleasaliva products of interest remain on a collection means following theelution protocols to obtain FS-1, FS-2 and FS-3 flea saliva extracts. Itis also within the scope of the invention that a formulation of thepresent invention can include flea saliva products removed from acollection means by eluting using other techniques, for example, byusing higher concentrations of eluants.

In another embodiment, a formulation of the present invention includesat least a portion of an ectoparasite saliva protein homologuepreferably having at least about 50 percent, more preferably at leastabout 75 percent, and even more preferably at least about 85 percentamino acid homology (identity within comparable regions) with at least aportion of at least one product contained in the saliva extracts FS-1,FS-2 or FS-3. Preferred homologues include at least a portion of anectoparasite saliva product having at least about 50 percent, morepreferably at least about 75 percent, and even more preferably at leastabout 85 percent amino acid homology with at least a portion of one ormore of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF,fspG1 fspG2, fspG3, fspH, fspI, fspJ1, fspj2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and fspN3. As such, also included are proteinshaving at least a portion of one of the following amino acid sequences:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 SEQID NO:33, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54 and/orSEQ ID NO:56.

In a preferred embodiment, a formulation of the present inventionincludes at least a portion of an ectoparasite saliva product homologueof the present invention that is encoded by a nucleic acid moleculehaving at least about 50 percent, more preferably at least about 75percent, and even more preferably at least about 85 percent homologywith a nucleic acid molecule encoding at least a portion of a productcontained in the saliva extracts FS-1, FS-2 or FS-3. A preferredectoparasite saliva product homologue is encoded by a nucleic acidmolecule having at least about 50 percent, more preferably at leastabout 75 percent, and even more preferably at least about 85 percent,homology with a nucleic acid molecule encoding at least a portion of oneor more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE,fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3.

In yet another embodiment, a formulation of the present inventionincludes a protein which, when digested with Endoproteinase Asp-N,generates proteolytic fragments that, when subjected to reverse phaseHPLC, migrate with peaks as depicted in FIG. 3. The reverse phase HPLCwas performed using the methods disclosed by Stone et al., EnzymaticDigestion of Proteins and HPLC Peptide Isolation, in A Practical Guideto Protein and Peptide Purification for Microsequencing, PT Matsudairaed., Academic Press, San Diego, Calif. (i.e., Narrowbore procedure:vydac C18 reverse-phase, 300 A, 5 μm support; flow rate of 0.2 ml/min;Solvent A being 0.6% TFA in water and Solvent B being 0.052% TFA in 80%acetonitrile in water; the sample was injected at 2% B; the gradientafter a hold at 2% B was 2-37.5% B over 60 min., 37.5%-75% B over 30min., 75%-98% B over 15 min.; and detection at 214 nm). An example ofsuch a protein is fspH, which also has the characteristics of amolecular weight of about 8613±6 daltons when determined by ESMS. Aparticularly preferred formulation of the present invention includes afspH protein having the amino acid sequence represented by SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:14 or SEQ ID NO:33.

In a preferred embodiment, a formulation of the present invention caninclude at least one isolated protein having (i.e., including) at leasta portion of the amino acid sequence (using the standard one letteramino acid code):

     Y G K Q Y S E K G G R G Q R H Q I L K K G K     Q Y S           S K       I     L   D   L      S      R (SEQ IDNO:1; representing a partial N-terminal sequence of fspA).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D R R V S K T C Q S G G K I Q S E X Q V V I K S G Q     H/Y I L E N Y X S D G R N N N N P C H L F C M R E C     R S G N G G C G N G G R T R P D S K H C Y C E A P Y      S (SEQ IDNO:2, representing the N-terminal nearly complete sequence of fspH).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D R R V S K T X Q S G G K I Q S E X Q V V I K S G Q     H/Y I L E N Y X S D G R (SEQ ID NO:14, representing a partialN-terminal sequence of fspH).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D S K H C Y C E A P Y S (SEQ ID NO:3; representing a partialN-terminal sequence of fspHe).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D G R N N N N P C H L F C M R E C R S G N G G C G N     G G R T R P D S K H C (SEQ ID NO:4; representing a partialN-terminal sequence of fspHh).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D R R V S K T C Q S G (SEQ ID NO:5; representing a partialN-terminal sequence of fspHj).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     E D I W K V N K K X T S G G K N Q D R K L D Q I I Q     K G Q Q V X X Q N X X K (SEQ ID NO:6; representing a partialN-terminal sequence of fspI).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     N S H E P G N T R K I R E V M D K L R K Q H P (SEQ ID NO:7;representing a partial N-terminal sequence of fspJ1).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     E I K R N S H E P G N T R K I R E V M D K L R K Q H P (SEQ ID NO:8;representing a partial N-terminal sequence of fspJ2).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     N D K E P G N T R K I R E V M D K L R K Q A Q P R T     D G Q R P K T X I M (SEQ ID NO:9; representing a partial N-terminalsequence of fspL1).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     X L X R N D K E P G N T R K I R E V M D K (SEQ ID NO:10;representing a partial N-terminal sequence of fspL2).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     N D E L K F V F V M A K (SEQ ID NO:11; representing a partialN-terminal sequence of fspN1).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     X D E L K F V F V M A K G P S X Q A X D Y P C (SEQ ID NO:12;representing a partial N-terminal sequence of fspN2).Note that although fspN1 and fspN2 appear to have similar, if notidentical, partial N-terminal sequences, the two proteins migratedifferently when submitted to Tris glycine SDS-PAGE, suggesting thatthey are different proteins, possibly due to a carboxyl-terminaltruncation of one of the proteins and/or post-translation modification,such as glycosylation.

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     E L K F V F A T A R G M S H T P C D Y P (SEQ ID NO:13; representinga partial N-terminal sequence of fspN3).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     S G K Q Y S E X G K      Q               S (SEQ ID NO:27;representing a partial N-terminal amino acid sequence of fspB).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     D R R V S K (SEQ ID NO:28; representing a partial N-terminal aminoacid sequence of fspG1).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     S K M V T E K X K S G G N N P S T K E V S I P (SEQ ID NO:29;representing a partial N-terminal amino acid sequence of fspG2).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     E V S I P S G K L T I E D F X I G N H Q (SEQ ID NO:30; representinga partial N-terminal amino acid sequence of fspG3).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     F S L C V L Y Q I V V A D R R V S K T C Q S G G K I     Q S E E/X Q V V I K S G Q H/Y I L E N Y X S D G R N     N N N P C H L F C M R E C R S G N G G C G N G G R T      R P D S(SEQ ID NO:33; representing a translation product of the nucleic acidsequence represented by SEQ ID NO:32, corresponding to fspH).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

     L T S G G K N Q D R K L D Q I I Q K G Q Q V K I Q N     I C K L I R D K P H T N Q E K E K C M K F C T K N V     C K G Y R G A C D G N I C Y C S R P S N L G P D W K     V N E R I E R L P I T K I L V S G N S S I S T T I T     N S K Y F E T K N S E T N E D S K S K K H S K E K C     R G G N D R G C D G N V L L L S T K K (SEQ ID NO:25; representing atranslation of the nucleic acid sequence represented by SEQ ID NO:24,corresponding to fspI).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

E D I W K V N K K L T S G G K N Q D R K L D Q I I Q K G Q Q V K I Q N IC K L I R D K P H T N Q E K E K C M K F C T K N V C K G Y R G A C D G NI C Y C S R P S N L G P D W K V N E R I E R L P I T K I L V S G N S S IS T T I T N S K Y F E T K N S E T N E D S K S K K H S K E K C R G G N DR G C D G N V L L L S T K K (SEQ ID NO:26; representing an apparentfull- length translation product of fspI).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

W K V N K K L T S G G K N Q D R K L D Q I I Q K G Q Q V K I Q N I C K LI R D K P H T N Q E K E K C M K F C T K N V C K G Y R G A C D G N I C YC S R P S N L G P D W K V N E R I E R L P I T K I L V S G N S S I S T TI T N S K Y F E T K N S E T N E D S K S K K H S K E K C R G G N D R G CD G N V L L L S T K K (SEQ ID NO:35; representing a translation productof the nucleic acid sequence represented by SEQ ID NO:34, correspondingto fspI).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

D I E N I K K G E G Q P G A P G G K E N N L S V L (SEQ ID NO:31;representing a partial N-terminal amino acid sequence of anEndoproteinase Asp-N fragment of fspN, named PfspN(100-101)).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

A R A R S V G S M K N K L K S F S E K Y V W A A L T S N D N L R K M S GG R M I N D I L N D I D N I K K G N G Q P N A P G K T E N K L S V S D RS S R Y L S S I R F S L F R P R Y K I E N Q D L E P S S L Y P G Q G A LH V I E L H K D K N Q W N V K T L Y R N N D Q Q E L K P M K L A K C G DT C S Y E T F K S T L Q S Y N M D K T A H D K L C K S S (SEQ ID NO:51;representing a translation product of the nucleic acid sequencerepresented by SEQ ID NO:50, corresponding to a fspN protein).Comparison of amino acid sequence SEQ ID NO:51 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:51 is about 28% identicalwith human prostatic acid phosphatase.

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

G T R K N E L K S F S E E Y L W R A L T S N E N L R K M S G G R M I N DI L N D I D S I K E E R D N R V L L E K Q E I K L S M L T V P Q A I L AA F V S A F A P K G T K I E N Q D L G P S S L Y P G Q G A L H V I E L HK D N N Q W S V K V L Y R N N D K M E L E P M K L P S C D D K C P C E LL N Q L Y N P M I (SEQ ID NO:53; representing a translation product ofthe nucleic acid sequence represented by SEQ ID NO:52, corresponding toa fspN protein).Comparison of amino acid sequence SEQ ID NO:53 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:53 is about 30% identicalwith human prostatic acid phosphatase.

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequence:

M W R L L L V I S S A L I I Q N V N A E L K F V F A T A T R Y V S H T PS P C D P G G P K I T N K P G D F Q R V (SEQ ID NO:54; representing apartial N-terminal amino acid sequence of a fspN protein).

A formulation of the present invention can also include at least oneisolated protein having at least a portion of the amino acid sequenceSEQ ID NO:56, representing an apparent full-length translation productof the nucleic acid sequence represented by SEQ ID NO:55, apparentlycorresponding to fspN3 protein. Comparison of amino acid sequence SEQ IDNO:56 with amino acid sequences reported in GenBank indicates that SEQID NO:56 is about 30% identical with human prostatic acid phosphatase.

It is to be appreciated that ectoparasite saliva proteins of the presentinvention include, but are not limited to, full-length proteins, hybridproteins, fusion proteins, multivalent proteins, and proteins that aretruncated homologues of, or are proteolytic products of, at least aportion of a protein contained in the saliva extracts FS-1, FS-2 orFS-3; and preferably at least a portion of saliva protein fspA, fspB,fspC1, fspC2, fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI,fspJ1, fspj2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and/orfspN3. As such, also included are proteins having at least a portion ofone of the following amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54 and/or SEQ ID NO:56. Asused herein, the term hybrid protein refers to a single protein producedfrom two different proteins.

The foregoing SEQ ID NO's represent amino acid sequences deducedaccording to methods disclosed in the Examples. It should be noted thatsince amino acid sequencing technology is not entirely error-free, theforegoing SEQ ID NO's, at best, represent an apparent amino acidsequence of the ectoparasite saliva proteins of the present invention.In addition, the variation seen in the foregoing SEQ ID NO's can also bedue, at least in part, to allelic variation since the proteins beingsequenced were derived from populations of fleas.

According to the present invention, a formulation of the presentinvention can include flea saliva proteins that have undergonepost-translational modification. Such modification can include, forexample, glycosylation. Glycosylation can include addition of N-linkedand/or O-linked oligosaccharides. It is to be appreciated thatpost-translational modification of a protein of the present inventioncan contribute to an epitope's ability to induce an allergic responseagainst the protein in an immediate or delayed hypersensitivityresponse.

Another embodiment of the present invention is an isolated nucleic acidmolecule capable of hybridizing, under stringent conditions, with anectoparasite saliva protein gene encoding an ectoparasite saliva proteinof the present invention. In accordance with the present invention, anisolated nucleic acid molecule is a nucleic acid molecule that has beenremoved from its natural milieu (i.e., that has been subject to humanmanipulation). As such, “isolated” does not reflect the extent to whichthe nucleic acid molecule has been purified. An isolated nucleic acidmolecule can include DNA, RNA, or derivatives of either DNA or RNA.

An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase “at least a portion of” an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with the correspondinggene under stringent hybridization conditions. An isolated nucleic acidmolecule of the present invention can also be produced using recombinantDNA technology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis. Isolated ectoparasite saliva proteinnucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications do not substantially interfere with the nucleicacid molecule's ability to encode an ectoparasite saliva protein of thepresent invention or to form stable hybrids under stringent conditionswith natural nucleic acid molecule isolates.

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one ectoparasite salivaprotein of the present invention, examples of such proteins beingdisclosed herein. Although the phrase “nucleic acid molecule” primarilyrefers to the physical nucleic acid molecule and the phrase “nucleicacid sequence” primarily refers to the sequence of nucleotides on thenucleic acid molecule, the two phrases can be used interchangeably,especially with respect to a nucleic acid molecule, or a nucleic acidsequence, being capable of encoding an ectoparasite saliva protein. Asheretofore disclosed, ectoparasite saliva proteins of the presentinvention include, but are not limited to, proteins having full-lengthectoparasite saliva protein coding regions, portions thereof, and otherectoparasite saliva protein homologues.

It is to be appreciated that an ectoparasite saliva protein of thepresent invention can be encoded by a full-length nucleic acid sequencewhich encodes a polyprotein. The polyprotein can be post-translationallyprocessed into multiple proteins which are found in saliva. As usedherein, an ectoparasite saliva protein gene includes all nucleic acidsequences related to a natural ectoparasite saliva protein gene such asregulatory regions that control production of an ectoparasite salivaprotein encoded by that gene (such as, but not limited to,transcription, translation or post-translation control regions) as wellas the coding region itself. A nucleic acid molecule of the presentinvention can be an isolated natural ectoparasite saliva protein nucleicacid molecule or a homologue thereof. A nucleic acid molecule of thepresent invention can include one or more regulatory regions,full-length or partial coding regions, or combinations thereof. Theminimal size of an ectoparasite saliva protein nucleic acid molecule ofthe present invention is the minimal size capable of forming a stablehybrid under stringent hybridization conditions with a correspondingnatural gene.

An ectoparasite saliva protein nucleic acid molecule homologue can beproduced using a number of methods known to those skilled in the art(see, for example, Sambrook et al., ibid.). For example, nucleic acidmolecules can be modified using a variety of techniques including, butnot limited to, classic mutagenesis techniques and recombinant DNAtechniques, such as site-directed mutagenesis, chemical treatment of anucleic acid molecule to induce mutations, restriction enzyme cleavageof a nucleic acid fragment, ligation of nucleic acid fragments,polymerase chain reaction (PCR) amplification and/or mutagenesis ofselected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid (e.g., the ability of a homologue to elicit an allergicresponse in animals having allergic dermatitis or the ability of ahomologue to act as an anti-coagulant) and/or by hybridization withisolated ectoparasite saliva protein nucleic acids under stringentconditions.

One embodiment of the present invention is an ectoparasite salivaprotein nucleic acid molecule capable of encoding at least a portion ofa flea saliva product, or a homologue thereof (e.g., saliva products ofother ectoparasites), contained in the saliva extracts FS-1, FS-2 orFS-3, wherein FS-1, when submitted to HPLC, resolves into peak A, peakB, peak C, peak D, peak E, peak F, peak G, peak H, peak I, peak J, peakK, peak L, peak M and/or peak N. A preferred nucleic acid molecule iscapable of encoding at least a portion of one or more of the proteinsfspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3,fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2and fspN3, or homologues thereof. As such, preferred nucleic acidmolecules include, but are not limited, nucleic acid molecules thatencode proteins having at least a portion of one or more of thefollowing amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:54 and/or SEQ ID NO:56, or homologuesthereof.

A preferred nucleic acid molecule of the present invention is capable ofhybridizing under stringent conditions to a nucleic acid that encodes atleast a portion of a flea saliva product, or a homologue thereof (e.g.,saliva products of other ectoparasites), contained in the salivaextracts FS-1, FS-2 or FS-3. Also preferred is an ectoparasite salivaprotein nucleic acid molecule that includes a nucleic acid sequencehaving at least about 65 percent, preferably at least about 75 percent,more preferably at least about 85 percent, and even more preferably atleast about 95 percent homology with the corresponding region(s) of thenucleic acid sequence encoding at least a portion of a flea salivaproduct, or a homologue thereof (e.g., saliva products of otherectoparasites), contained in the saliva extracts FS-1, FS-2 or FS-3. Aparticularly preferred nucleic acid sequence is a nucleic acid sequencehaving at least about 65 percent, preferably at least about 75 percent,more preferably at least about 85 percent, and even more preferably atleast about 95 percent homology with a nucleic acid sequence encoding atleast a portion of one or more of the proteins fspA, fspB, fspC1, fspC2,fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspj2,fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3. As such, alsopreferred are nucleic acid molecules having at least about 65 percent,preferably at least about 75 percent, more preferably at least about 85percent, and even more preferably at least about 95 percent homologywith a nucleic acid sequence encoding at least a portion of one or moreof the following amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:54 and/or SEQ ID NO:56.

Such nucleic acid molecules can be a full-length gene and/or a nucleicacid molecule encoding a full-length protein, a hybrid protein, a fusionprotein, a multivalent protein or a truncation fragment. More preferrednucleic acid molecules of the present invention comprise isolatednucleic acid molecules having a nucleic acid sequence as represented bySEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:50,SEQ ID NO:52 or SEQ ID NO:55. SEQ ID NO:20, a nucleic acid sequence thatincludes about 60 nucleotides of the apparent gene encoding flea salivaprotein fspH, includes about 25 percent of the coding region of fspH.SEQ ID NO:24, a nucleic acid sequence that includes about 573nucleotides of the apparent gene encoding flea saliva protein fspI,encodes a protein of about 149 amino acids, represented by SEQ ID NO:25.The entire translation product of fspI is apparently about 158 aminoacids and is denoted SEQ ID NO:26. SEQ ID NO:32, a 242 bp nucleic acidsequence of the apparent gene encoding flea saliva protein fspH, encodesa protein of about 80 amino acids, which is denoted SEQ ID NO:33. SEQ IDNO:34, a 591 bp nucleic acid sequence of the apparent gene encoding fleasaliva protein fspI, encodes a protein of about 155 amino acids, whichis denoted SEQ ID NO:35. SEQ ID NO:50, a 646 bp nucleic acid sequence ofthe apparent gene encoding a fspN flea saliva protein, encodes a proteinof about 172 amino acids, which is denoted SEQ ID NO:51. SEQ ID NO:52, a612 bp nucleic acid sequence of the apparent gene encoding a fspN fleasaliva protein, encodes a protein of about 153 amino acids, which isdenoted SEQ ID NO:53. SEQ ID NO:55, a 1197 bp nucleic acid sequence ofthe apparent gene encoding fspN3, encodes a protein of about 398 aminoacids, which is denoted SEQ ID NO:56.

Knowing a nucleic acid molecule of an ectoparasite saliva protein of thepresent invention allows one skilled in the art to make copies of thatnucleic acid molecule as well as to obtain a nucleic acid moleculeincluding additional portions of ectoparasite saliva protein-encodinggenes (e.g., nucleic acid molecules that include the translation startsite and/or transcription and/or translation control regions), and/orectoparasite saliva protein nucleic acid molecule homologues. Knowing aportion of an amino acid sequence of an ectoparasite saliva protein ofthe present invention allows one skilled in the art to clone nucleicacid sequences encoding such an ectoparasite saliva protein. Inaddition, a desired ectoparasite saliva protein nucleic acid moleculecan be obtained in a variety of ways including screening appropriateexpression libraries with antibodies which bind to ectoparasite salivaproteins of the present invention; traditional cloning techniques usingoligonucleotide probes of the present invention to screen appropriatelibraries or DNA; and PCR amplification of appropriate libraries, or RNAor DNA using oligonucleotide primers of the present invention (genomicand/or cDNA libraries can be used). To isolate flea saliva proteinnucleic acid molecules, preferred cDNA libraries include cDNA librariesmade from unfed whole flea, fed whole flea, fed flea midgut, unfed fleamidgut, and flea salivary gland. Techniques to clone and amplify genesare disclosed, for example, in Sambrook et al., ibid. The Examplessection includes examples of the isolation of cDNA sequences encodingflea saliva proteins of the present invention.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of aflea saliva product, or a homologue thereof (e.g., saliva products ofother ectoparasites), contained in the saliva extracts FS-1, FS-2 orFS-3. A preferred oligonucleotide is capable of hybridizing, understringent conditions, with a nucleic acid molecule that is capable ofencoding at least a portion of one or more of the proteins fspA, fspB,fspC1, fspC2, fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI,fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3,or homologues thereof. As such, certain preferred oligonucleotides arecapable of hybridizing to a nucleic acid molecule capable of encoding aprotein having at least a portion of one or more of the following aminoacid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:54 and/or SEQ ID NO:56, or homologues thereof. Certainpreferred oligonucleotides are capable of hybridizing to nucleic acidmolecules including nucleic acid sequences represented by SEQ ID NO:20,SEQ ID:24, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:52, SEQID NO:55 or complements thereof.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimal size of such oligonucleotides is thesize required to form a stable hybrid between a given oligonucleotideand the complementary sequence on another nucleic acid molecule of thepresent invention. Minimal size characteristics are disclosed herein.The size of the oligonucleotide must also be sufficient for the use ofthe oligonucleotide in accordance with the present invention.Oligonucleotides of the present invention can be used in a variety ofapplications including, but not limited to, as probes to identifyadditional nucleic acid molecules, as primers to amplify or extendnucleic acid molecules or in therapeutic applications to inhibit, forexample, expression of saliva proteins by ectoparasites. Suchtherapeutic applications include the use of such oligonucleotides in,for example, antisense-, triplex formation-, ribozyme- and/or RNAdrug-based technologies. The present invention, therefore, includes sucholigonucleotides and methods to interfere with the production ofectoparasite saliva proteins by use of one or more of such technologies.

The present invention also includes a recombinant vector, which includesan ectoparasite saliva protein nucleic acid molecule of the presentinvention inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to ectoparasite saliva protein nucleic acidmolecules of the present invention. The vector can be either RNA or DNA,either prokaryotic or eukaryotic, and typically is a virus or a plasmid.Recombinant vectors can be used in the cloning, sequencing, and/orotherwise manipulating of ectoparasite saliva protein nucleic acidmolecules of the present invention. One type of recombinant vector,herein referred to as a recombinant molecule and described in moredetail below, can be used in the expression of nucleic acid molecules ofthe present invention. Preferred recombinant vectors are capable ofreplicating in the transformed cell.

A preferred nucleic acid molecule to include in a recombinant vector ofthe present invention is a nucleic acid molecule that encodes at least aportion of at least one flea saliva product, or a homologue thereof(e.g., saliva products of other ectoparasites), contained in the salivaextracts FS-1, FS-2 or FS-3. A particularly preferred nucleic acidmolecule to include in a recombinant vector is capable of encoding atleast a portion of one or more of the proteins fspA, fspB, fspC1, fspC2,fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2,fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, or homologuesthereof. As such, also included are nucleic acid molecules that encode aprotein having at least a portion of one or more of the following aminoacid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:54 and/or SEQ ID NO:56, or homologues thereof, andnucleic acid molecules including at least a portion of a nucleic acidsequence represented by SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:50, SEQ ID NO:52 and/or SEQ ID NO:55.

In one embodiment, an isolated ectoparasite saliva protein of thepresent invention is produced by culturing a cell capable of expressingthe protein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell that is capable of expressing the ectoparasite saliva protein, therecombinant cell being produced by transforming a host cell with one ormore nucleic acid molecules of the present invention. Transformation ofa nucleic acid molecule into a cell can be accomplished by any method bywhich a nucleic acid molecule can be inserted into the cell.Transformation techniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a host cell include nucleic acid molecules that encode atleast a portion of a flea saliva product, or a homologue thereof (e.g.,saliva products of other ectoparasites), contained in the salivaextracts FS-1, FS-2 or FS-3. Particularly preferred nucleic acidmolecules with which to transform a host cell are as disclosed hereinfor including in recombinant vectors of the present invention.

Suitable host cells to transform include any cell that can betransformed and that can express the introduced ectoparasite salivaprotein. Such cells are, therefore, capable of producing ectoparasitesaliva proteins of the present invention after being transformed with atleast one nucleic acid molecule of the present invention. Host cells canbe either untransformed cells or cells that are already transformed withat least one nucleic acid molecule. Suitable host cells of the presentinvention can include bacterial, fungal (including yeast), insect,animal and plant cells. Preferred host cells include bacterial, yeast,insect and mammalian cells, with bacterial (e.g., E. coli) and insect(e.g., Spodoptera) cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth, insect and mammalian cells, such as, butnot limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB,bacteriophage lambda (λ) (such as λp_(L) and λp_(R) and fusions thatinclude such promoters), bacteriophage T7, T7lac, bacteriophage T3,bacteriophage SP6, bacteriophage SP01, metallothionein, alpha matingfactor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such asSindbis virus subgenomic promoters), baculovirus, Heliothis zea insectvirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus40, retrovirus actin, retroviral long terminal repeat, Rous sarcomavirus, heat shock, phosphate and nitrate transcription control sequencesas well as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins). Transcription control sequences of thepresent invention can also include naturally occurring transcriptioncontrol sequences naturally associated with a DNA sequence encoding anectoparasite saliva protein.

Expression vectors of the present invention may also contain secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed ectoparasite saliva protein to be secreted from the cell thatproduces the protein. Suitable signal segments include an ectoparasitesaliva protein signal segment or any heterologous signal segment capableof directing the secretion of an ectoparasite saliva protein, includingfusion proteins, of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA),interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of an ectoparasite nucleic acid molecule of thepresent invention can enhance the stability during production, storageand/or use of the protein encoded by the nucleic acid molecule.Furthermore, a fusion segment can function as a tool to simplifypurification of an ectoparasite saliva protein, such as to enablepurification of the resultant fusion protein using affinitychromatography. A suitable fusion segment can be a domain of any sizethat has the desired function (e.g., increased stability and/orpurification tool). It is within the scope of the present invention touse one or more fusion segments. Fusion segments can be joined to aminoand/or carboxyl termini of an ectoparasite saliva protein. Linkagesbetween fusion segments and ectoparasite saliva proteins can beconstructed to be susceptible to cleavage to enable straight-forwardrecovery of the ectoparasite saliva proteins. Fusion proteins arepreferably produced by culturing a recombinant cell transformed with afusion nucleic acid sequence that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of anectoparasite saliva protein.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed. A preferred recombinantmolecule includes one or more nucleic acid molecules that encode atleast a portion of a flea saliva product, or a homologue thereof (e.g.,saliva products of other ectoparasites), contained in the salivaextracts FS-1, FS-2, or FS-3. Particularly preferred nucleic acidmolecules to include in a recombinant molecule are as disclosed hereinfor including in a recombinant vector of the present invention.

A recombinant cell of the present invention includes any cellstransformed with at least one of any nucleic acid molecules of thepresent invention. A preferred recombinant cell is a cell transformedwith at least one nucleic acid molecule that encodes at least a portionof a flea saliva product, or a homologue thereof (e.g., saliva productsof other ectoparasites), contained in the saliva extracts FS-1, FS-2and/or FS-3. A preferred recombinant cell is transformed with at leastone nucleic acid molecule that is capable of encoding at least a portionof one or more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2,fspE, fspf, fspG1 fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1,fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, or homologues thereof. Assuch, also included are nucleic acid molecules that encode a proteinhaving at least a portion of one or more of the following amino acidsequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:54 and/or SEQ ID NO:56, or homologues thereof, and nucleicacid molecules including at least a portion of a nucleic acid sequencerepresented by SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:50, SEQ ID NO:52 and/or SEQ ID NO:55. Particularly preferredrecombinant cells include E. coli transformed with at least one of theaforementioned nucleic acid molecules.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

In accordance with the present invention, recombinant cells can be usedto produce an ectoparasite saliva protein of the present invention byculturing such cells under conditions effective to produce such aprotein, and recovering the protein. Effective conditions to produce aprotein include, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing anectoparasite saliva protein. Such a medium is typically an aqueousmedium comprising assimilable carbohydrate, nitrogen and phosphatesources, as well as appropriate salts, minerals, metals and othernutrients, such as vitamins. The medium may comprise complex nutrientsor may be a defined minimal medium.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantectoparasite saliva proteins may either remain within the recombinantcell; be secreted into the fermentation medium; be secreted into a spacebetween two cellular membranes, such as the periplasmic space in E.coli; or be retained on the outer surface of a cell or viral membrane.The phrase “recovering the protein” refers simply to collecting thewhole fermentation medium containing the protein and need not implyadditional steps of separation or purification. Ectoparasite salivaproteins of the present invention can be purified using a variety ofstandard protein purification techniques, such as, but not limited to,affinity chromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, chromatofocusing anddifferential solubilization.

Ectoparasite saliva proteins are preferably retrieved in “substantiallypure” form. As used herein, “substantially pure” refers to a purity thatallows for the effective use of the protein as a therapeutic compositionor diagnostic. For example, an animal being administered dosages ofectoparasite saliva protein isolated from a recombinant cell of thepresent invention should exhibit no substantial toxicity fromcontaminants mixed with the protein.

Ectoparasite saliva products substantially free of contaminatingmaterial can be isolated using a saliva collection apparatus of thepresent invention. A saliva collection apparatus of the presentinvention is designed to stimulate (i.e., cause) ectoparasites retainedin the container to feed, and thereby to release saliva, which iscollected separate from contaminating material.

Ectoparasites attach and feed from warm-blooded host animals. A hostanimal, as used herein, refers to an animal that ectoparasites can feedfrom or on. Without being bound by theory, it is believed thatectoparasites, such as fleas, possess heat receptors which enables theectoparasite to sense a temperature differential between the warm skinof the host and the ambient air. The temperature differential stimulates(i.e., causes) the ectoparasite to feed from the warm surface (i.e.,from the warm animal skin). It is also believed that motion, vibrationand darkness can be sensed by ectoparasites, thereby encouraging them tofeed. An ectoparasite feeds by penetrating the dermis of an animal withits mouthparts, the mouthparts remaining in that position while theectoparasite secretes saliva to enhance feeding. During feeding, anectoparasite can release contaminants such as blood proteins and fecalmaterial.

A saliva collection apparatus of the present invention includes achamber and housing such that a temperature differential between thechamber and housing of the apparatus is maintained which causes orpromotes ectoparasites retained in the housing to attempt to feed fromthe chamber. When ectoparasites housed in such an apparatus attempt tofeed in accordance with the present invention, the arthropods releasesaliva which is collected in such a manner that proteins, and otherproducts, in the saliva are isolated substantially free of contaminatingmaterial. In order to collect saliva substantially free of contaminatingmaterial, an apparatus of the present invention also includes acollection means to capture saliva on a surface separate from thesurface which captures the contaminating material.

A saliva collection apparatus of the present invention can be used tocollect saliva from any ectoparasite such as those disclosed herein.Ectoparasites of the present invention can feed on any animalsusceptible to ectoparasite infestation (i.e., a host animal), includingbut not limited to, a wide variety of vertebrates. Preferred hostanimals include mammals and birds. More preferred host animals includecats, dogs, humans, horses, rabbits, sheep, cattle, swine, goats,raccoons, ferrets, rats and opossums as well as other pets, economicfood animals and animals that are hosts for fleas that infest pets andeconomic food animals. Particularly preferred host animals are cats anddogs.

Particularly preferred ectoparasites of the present invention from whichto collect saliva include any suitable species of flea. Preferred fleasinclude fleas capable of infesting cats and dogs. Newly hatched fleas(i.e., recently emerged from a pupal state) that have not had a firstblood meal are preferred for the following reasons: Because newlyemerged fleas have not had a first blood meal, such fleas attempt tofeed. Since newly emerged fleas have not had a blood meal, they also donot release as much contaminating material as do fed fleas. Newlyemerged fleas live longer without a blood meal than do fleas which havehad at least one blood meal. It should be noted that fed fleas can alsobe used with an apparatus of the present invention.

It will be obvious to one of skill in the art that a saliva collectionapparatus of the present invention is useful for collecting saliva fromany ectoparasite. For the purpose of illustration, a flea salivacollection apparatus of the present invention is described in detailbelow. Such description is not intended, in any way, to limit the scopeof the present invention. It is within the skill of one in the art tocollect saliva from other ectoparasites in a straightforward mannerbased on methods to collect saliva from fleas.

One embodiment of the present invention is a saliva collection apparatusthat includes a chamber and a housing operatively connected to aninterface in such a manner that a temperature differential is maintainedbetween the chamber and the housing. The interface includes a collectionmeans and a barrier means positioned such that, in order to attempt tofeed, flea mouthparts penetrate the barrier means prior to thecollection means. The temperature differential between the chamber andthe housing is a difference in temperature suitable to attract fleasretained in the housing to attempt to feed through the interface and,thereby deposit saliva products on the collection means. Due to therelative positioning of the collection means and the barrier means,contaminating material is deposited on the barrier means.

A flea saliva collection apparatus of the present invention includes ahousing. A housing can comprise any material capable of retaining fleasthat provides structural support and that can be connected to aretaining means. The housing is preferably made of a material capable ofwithstanding cleaning and/or sterilization procedures commonly used bythose skilled in the art. As such, the housing can be reused. Preferredhousing materials of the present invention include, but are not limitedto, plastic, metal, rubber, wood and glass materials and combinations ofsuch materials. More preferred housing materials include plastic andmetal materials with plastic materials being even more preferred.Preferred plastic materials include plexiglass, teflon, nylon andpolycarbonate. A particularly preferred plastic material is plexiglass,or other durable, break-resistant plastic, preferably clear so as toallow viewing of fleas inside the container.

In accordance with the present invention, the size of a housing of thepresent invention is such that the housing can support a desired numberof fleas without overcrowding. Both surface area and the volume of thehousing can be important. The size of the housing can vary according tothe number of fleas to be retained in the housing. Preferably, the sizeof the housing is sufficient to maintain from about 1,000 fleas to about6,000 fleas per housing for about 72 hours, more preferably from about2,000 fleas to about 5,000 fleas per housing for about 72 hours, andeven more preferably from about 3,000 fleas to about 4,000 fleas perhousing for about 72 hours.

A suitable height for a housing of the present invention is a heightthat is sufficiently high to allow room for fleas to move about whilefeeding. The height of a housing for fleas is preferably from about 1.0centimeters (cm) to about 3.0 cm, more preferably from about 1.5 cm toabout 2.5 cm, and even more preferably from about 1.8 cm to about 2.2cm.

The shape of a housing of the present invention can be any shape havingat least one flat surface suitable for feeding by fleas contained withinthe housing. A housing of the present invention is preferably shaped asa cylinder, a box having four or more sides, a half-dome, or a halfcylinder. A particularly preferred shape is a short cylinder.

The diameter of a preferred housing of the present invention can varywidely. Different diameter containers can be used according to, forexample, the number of fleas to be placed into the housing withoutovercrowding. The interior diameter of a rounded housing of the presentinvention is preferably from about 4.0 cm to about 5.5 cm, morepreferably from about 4.5 cm to about 5.5 cm, and even more preferablyabout 5.0 cm.

According to the present invention, the size, shape, height, anddiameter of the housing can vary for different ectoparasites dependingupon the size and number of arthropods retained in the housing.

In accordance with the present invention, a housing is operativelyconnected to a retaining means and an exchange means. As used herein,“operatively connected” refers to combining portions of a salivacollection apparatus of the present invention in such a manner thatfleas can be retained within the apparatus and can deposit saliva on thecollection means. A retaining means of the present invention ispenetrable by the mouthparts of fleas. A retaining means of the presentinvention can comprise any material or combination of materials that issuitable for retaining fleas and through which fleas can feed (i.e., theretaining means is penetrable by flea mouthparts). As such, theretaining means may comprise a material having openings sufficientlylarge (i.e., large enough) for flea mouthparts to penetrate, butsufficiently small (i.e., small enough) so as to effectively preventloss of any fleas retained therein. Preferred retaining means comprise amaterial having openings of from about 0.25 millimeters (mm) to about0.50 mm, more preferably having openings of from about 0.30 mm to about0.50 mm, and even more preferably having openings of from about 0.35 mmto about 0.45 mm. One of skill in the art will recognize that the sizeof the openings can vary according to the type of ectoparasite retainedin the housing of an apparatus. For example, maintenance of particularlysmall ectoparasites such as, but not limited to, lice may requireretaining means having smaller openings. Conversely, a retaining meansfor hard ticks, which are ectoparasites that cement their mouthpartsinto the host animal, require larger openings, preferably in the rangeof about 1 mm.

Preferred materials for use as retaining means include, but are notlimited to, metallic mesh, nylon mesh, plastic film, cloth andcombinations of such materials. More preferred retaining means includenylon mesh and metal mesh, and an even more preferred retaining meansincludes nylon mesh. The collection apparatus can be retrofitted with avariety of retaining means. Preferred retaining means are reusable.

An exchange means of the present invention can comprise any material orcombination of materials capable of maintaining a permissive environmentfor fleas within the housing by allowing the exchange of gas, humidityand heat between the interior environment of the housing and theenvironment exterior to the housing. The housing can be retrofitted withdifferent exchange means having different gas, humidity and heatpermeabilities. As used herein, the term gas refers to any atmosphericgases required for flea survival, including, but not limited to, carbondioxide, oxygen, and nitrogen. Gas can also refer to gaseous productsproduced by fleas while maintained in a housing of the presentinvention, such as gaseous products of metabolism including expirationsor gases from feces.

Exchange means of the present invention are comprised of materialshaving openings that are sufficiently large to allow gas, heat andhumidity to escape, but sufficiently small so as to effectively preventloss of fleas. Preferred exchange means comprise a material havingopenings of from about 0.10 millimeters (mm) to about 0.45 mm, morepreferably having openings of from about 0.10 mm to about 0.30 mm, andeven more preferably having openings of from about 0.13 mm to about 0.15mm.

Preferred materials to use as an exchange means include, but are notlimited to, metallic mesh, nylon mesh, plastic, cloth and combinationsof such materials. More preferred exchange means include nylon mesh,metal mesh, and combinations of such materials and an even morepreferred exchange means includes nylon mesh. Preferred exchangematerials are reusable.

In accordance with the present invention, an apparatus includes achamber operatively connected to a housing. A chamber of the presentinvention is capable of maintaining an internal temperature suitable tocreate a temperature differential between a housing and a chamber of anapparatus which promotes deposition of saliva by fleas retained in thehousing on a collection means of an apparatus. A preferred chamber isalso capable of maintaining an internal humidity level suitable for thesurvival of ectoparasites contained in the apparatus (e.g., suitable toprevent desiccation of the ectoparasites). A chamber of the presentinvention is also capable of being attached to an artificial feedingsystem as described in detail in the Examples. A chamber can compriseany material capable of maintaining suitable temperature and humiditylevels within the chamber. A chamber is preferably made of a materialcapable of withstanding cleaning or sterilization procedures commonlyused by those skilled in the art. As such, a chamber can be reused.Preferred chamber materials of the present invention include, but arenot limited to, glass, plastic, metal, rubber, and wood materials andcombinations of such materials. More preferred chamber materials includeglass and plastic materials with glass materials being even morepreferred.

In accordance with the present invention, the size of a chamber of thepresent invention is such that the chamber can maintain a suitabletemperature level to stimulate fleas to deposit saliva on the collectionmeans of the apparatus. The size of the chamber can vary according tothe amount of blotting material (as described in detail below) to beplaced in the chamber, the diameter of the collection means to beattached to the chamber or whether the chamber is to be attached to anartificial feeding system as described in detail in the Examples.Preferably, the height of a chamber of the present invention is highenough to allow a suitable amount of blotting material to be placed inthe chamber, such that the blotting material maintains a humidity levelin the chamber suitable for flea survival. The height of a chamber ispreferably from about 1.0 cm to about 7.0 cm, more preferably from about2.0 cm to about 6.0 cm, and even more preferably from about 3.0 cm toabout 5.0 cm.

In accordance with the present invention, the shape of a chamber can beany shape having at least one open end to which an interface of thepresent invention can be attached. A chamber of the present invention ispreferably shaped as a cylinder open at both ends or a cylinder open atone end. A particularly preferred shape is a cylinder open at both ends.

The diameter of a preferred chamber of the present invention can varywidely. Different diameter chambers can be used according to, forexample, the diameter of the interface to be attached to the chamber orthe diameter of the housing to be attached to the chamber. The interiordiameter of a chamber of the present invention is preferably from about2.0 cm to about 6.5 cm, more preferably from about 3.0 cm to about 5.5cm, and even more preferably from about 4.0 cm to about 4.5 cm.

A chamber of the present invention can contain a blotting means suitablefor maintaining a humidity level in the chamber suitable for fleasurvival. Methods for maintaining suitable humidity levels are describedin detail below. A chamber of the present invention can contain food orwater, but preferably is humid (i.e., damp but not wet) and does notcontain food.

A saliva collection apparatus of the present invention includes aninterface. An interface of the present invention includes means capableof collecting saliva products substantially free of contaminatingmaterial. As such, an interface of the present invention is penetrableby flea mouthparts but capable of keeping contaminating material, suchas blood and fecal material, separate from flea saliva products secretedby fleas as they attempt to feed. An interface of the present inventioncomprises a means for collecting saliva products and a means forcreating a barrier between contaminating material and collected salivaproducts.

A collection means of the present invention can be of any materialcapable of collecting (i.e., adsorbing) at least a portion of salivaproteins deposited (i.e., secreted) by retained fleas that areattempting to feed through the interface. In addition, a collectionmeans of the present invention is capable of collecting salivacomponents other than saliva proteins deposited by fleas attempting tofeed through the interface. The collection means is such that salivaproducts not only can bind to the collection means but also can beeluted (i.e., extracted) therefrom upon exposure to a suitable eluent(i.e., extractant). As such, preferred collection means materials of thepresent invention include materials that are hydrophobic and have a lowbinding capacity since saliva components are easily eluted from suchmaterial. The material of a collection means of the present inventionshould also be capable of being penetrated by the mouthparts of fleas.

Preferred collection means materials of the present invention include,but are not limited to, nylon, nitrocellulose, CM-derivatized,diethylaminoethyl (DEAE)-derivatized, paper, polysulfone, celluloseester, polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF)membranes. A particularly preferred anion exchange membrane collectionmeans material includes DE-81 chromatography paper, which can beobtained from Whatman, Inc., Clifton, N.J. Particularly preferredcollection means materials include PVDF. A preferred PVDF collectionmeans material includes Durapore™.

The shape of a collection means of the present invention can varyaccording to the shape of the chamber to which the collection means isto be attached. A preferred shape of a collection means includes, but isnot limited to, a round shape or a box-like shape having four or moresides, with a round shape being more preferred.

The size of a collection means of the present invention can also varyaccording to the size of the chamber to which the collection means is tobe attached. The size of a collection means is preferably larger thanthe open end of a chamber, thereby preventing the collection means frompassing into the chamber. The size of a collection means is preferablyfrom about 2.2 cm to about 6.5 cm in diameter, more preferably fromabout 3.2 cm to about 5.7 cm, and even more preferably from about 4.2 cmto about 4.7 cm.

A saliva collection apparatus of the present invention provides for anovel barrier means which enables collection of ectoparasite salivasubstantially free of contaminating material. A barrier means of thepresent invention can be any material capable of substantiallypreventing contaminating material from contacting the collection means(i.e., substantially prevents the passage of contaminating material suchas flea fecal material and blood products through the collection means),but is also capable of being penetrated by the mouthparts of fleas andof allowing the passage of saliva through the barrier means. Preferably,the thickness of a barrier means material of the present invention ismicrons thick. Preferred barrier means materials of the presentinvention include, but are not limited to, very thin plastic, teflon,cloth, paper, paraffin and wax materials. More preferred barrier meansmaterials of the present invention include stretched plastic, with SaranWrap™ and particularly Parafilm™, stretched very thin (i.e., as thin ascan be stretched by machine and/or hand), being even more preferred.

The size of a barrier means of the present invention can vary accordingto the size of the chamber to which the barrier means is to be attached.The size of the barrier means preferably is sufficiently large that thebarrier means can extend up the sides of a chamber of the presentinvention, thereby enabling the barrier means to be secured to thechamber. The size of the barrier means is sufficiently small such thatthe barrier means does not interfere with, for example, the ability ofthe saliva collection apparatus containing the chamber to be attached toan artificial feeding system.

According to the present invention, a collection means and a barriermeans are operatively connected to a chamber of a saliva collectionapparatus in such a manner that fleas retained in the housing of suchapparatus are capable of penetrating both the barrier means and thecollection means to deposit saliva on the collection means. A collectionmeans of the present invention preferably is removably attached to asite on a chamber by a barrier means. A preferred site of attachment ofa collection means and a barrier means is the portion of a chamberdesigned to interface with a housing. A more preferred site ofattachment of a collection means and a barrier means is the open end ofa chamber.

A saliva collection apparatus of the present invention can also includea blotting means. A blotting means of the present invention is capableof maintaining a humidity within the apparatus suitable for fleasurvival and, as such, is capable of retaining liquid for the period oftime fleas are retained in the apparatus. As such, a blotting means canbe any suitable absorbent material. Preferred blotting means includenatural and synthetic sponges, foam, paper, cloth, and agarose. Morepreferred blotting means material include sponges and paper, with filterpaper being even more preferred. In a particularly preferred embodiment,one or more pieces of VWR Blotting Pads #320 (available from VWRScientific, Denver, Colo.) comprise a blotting means.

As stated above, a saliva collection apparatus of the present inventionis capable of maintaining a temperature differential between a housingand a chamber of the apparatus. A suitable temperature differentialwithin an apparatus of the present invention includes a temperaturedifferential which stimulates fleas retained in the apparatus topenetrate the interface of the apparatus and deposit saliva on thecollecting means. Preferred temperatures in a chamber of the presentinvention range from about 20° C. to about 45° C., whereas preferredtemperatures in a housing of the present invention range from about 5°C. to about 35° C. In a preferred embodiment, the temperature in thechamber ranges from about 35° C. to about 40° C. and the temperature inthe housing ranges from about 10° C. to about 30° C. A particularlypreferred chamber temperature ranges from about 35° C. to about 37° C.;and a particularly preferred housing chamber temperature is from about20° C. to about 27° C.

The survival of ectoparasites can be affected by humidity. As such, thehumidity level in a housing of an apparatus of the present invention issuitable for maintaining the survival of ectoparasites retained therein.Suitable relative humidity levels within an apparatus of the presentinvention can vary depending upon the ectoparasite contained within theapparatus. As used herein, relative humidity refers to the degree ofatmospheric water vapor relative to the maximum degree of atmosphericwater vapor that results in precipitation. Thus, relative humidity isexpressed in percent humidity, wherein 100% humidity representssaturation of atmospheric water vapor. Preferred humidity levels in achamber of the present invention range from about 50% to about 100%,whereas preferred humidity levels in a housing of the present inventionrange from about 40% to about 60%. In a preferred embodiment, thehumidity levels in the chamber ranges from about 50% to about 94% andthe humidity level in the housing is about 50%.

Another embodiment of the present invention is the use of contrastingcolors to attract fleas. For example, at least one surface of acollection apparatus of the present invention can be of a colorsufficiently dark to attract fleas to penetrate the interface of theapparatus. Without being bound by theory, it is believed that fleas arecapable of sensing light from dark and preferably tend to feed towards adark surface. Therefore, according to the present invention, a chambercan be darker than a housing, thereby attracting fleas to the interfacebetween the chamber and the housing. Suitable dark colors include colorsranging from black to light brown, preferably black.

A preferred embodiment of a collection apparatus of the presentinvention is depicted in FIGS. 4A and 4B. A saliva collection apparatus(2) is separable into a housing (4) and a chamber (6). A cross-sectionof a saliva collection apparatus of the present invention (2) is shownin FIG. 4A. Referring to FIGS. 4A and 4B, the housing (4) has anopen-ended cylinder having a first portion (8) and a second portion(10). The second portion (10) of the housing (4) has an outer diameter(12) and an inner diameter (14). An exchange means (16) is operativelyattached to one end of the first portion (8) of the housing (4) and aretaining means (18) is attached at the opposite end of the firstportion (8), between the first portion (8), and the outer diameter (12)and inner diameter (14) of the second portion (10). The exchange means(16) and retaining means (18) are attached in a manner that preventsfleas from escaping. Means of attaching an exchange means (16) and aretaining means (18) to the first portion (8) of the housing (4)include, but are not limited to, rubber cement, glue, tape, solder andaraldite. A preferred means of attachment is rubber cement.

The chamber (6) of the saliva collection apparatus (2) has an open endedcylinder having a top end (20) and a bottom end (22). The top end (20)has a suitable diameter to enable the attachment of the chamber (6) toan artificial feeding system such as that described in detail in theExamples. The bottom end (22) has a suitable diameter such that thebottom end (22) can be reversibly attached to the housing (4) in such amanner as to provide a sliding fit. The bottom end (22) is covered by acollection means (24). The collection means (24) has a larger diameterthan the inner circumference of the bottom end (22) of the chamber (6),thereby preventing the collection means (24) from passing into the innerspace (26) of the chamber (6). Preferably, the diameter of thecollection means (24) is not greater than the outer circumference of thebottom end (22) of the chamber (6). The collection means (24) can beconnected to the bottom end (22) in order to provide a detachableconnection, thereby facilitating removal of the collection means (24)from the saliva collecting apparatus (2) to recover saliva products fromthe collection means (24).

A collection means (24) attached to the bottom end (22) of a chamber (6)is covered by a barrier means (28). The barrier means (28) isoperatively connected to a chamber (6) in such a manner that a seal isformed preventing contact of the collection means (24) by contaminatingmaterial deposited by fleas. The barrier means (28) can be connected tothe chamber (6) in order to provide a detachable connection. Preferably,the barrier means (28) comprises a stretchable plastic material, such asParafilm™, which is stretched as thin as possible across a collectionmeans (24) contacting the bottom end (22) of a chamber (6) and furtherstretched along the sidewall (30) of the chamber (6) towards the top end(20) of the chamber (6). The barrier means (28) can be further securedto the sidewall (30) of the chamber (6) using a rubber seal (32). Therubber seal (32) detachably connects the portion of the barrier means(28) which meets the sidewall (30) of the chamber (6), thereby furthersecuring the collection means (24) to the chamber (6) and seal in thechamber (6) environment.

Blotting (absorbent) material can be placed in the inner space (26) atthe bottom end (22) of a chamber (6) to form a blotting means (34). Theblotting means (34) can comprise one or more individual blotting pads(e.g., pieces of blotting material). Preferably, the blotting means (34)is from about 2.0 mm thick to about 15.0 mm thick (when dry) when placedin a 47 cm high chamber (6), more preferably is from about 2.2 mm thickto about 12.5 mm thick (when dry) when placed in a 47 mm high chamber(6), and even more preferably is from about 2.45 mm thick to about 10.0mm thick (when dry) when placed in a 47 mm high chamber (6). In aparticularly preferred embodiment, the blotting means (34) comprisesfrom about 2 to 6 pieces of VWR Blotting Pads #320, and preferably fromabout 3 to 5 pieces of VWR Blotting Pads #320. The diameter of theblotting means (34) is selected to contact the inner sidewall (36) ofthe chamber (6). The blotting means (34) is preferably sufficientlypre-wetted to provide humidity to the chamber (6) but not so wet thatliquid drips from the blotting means (34). The blotting means (34) isjuxtaposed to the side of the collection means (24) facing the top end(20) of the chamber (6). The blotting means (34) can directly contactthe collection means (24) in a detachable manner.

The chamber (6) is reversibly separable from the housing (4). Thechamber (6) can be interconnected to the housing (4) in any reversiblysecure manner such as sliding, snapping or screwing together.Preferably, the chamber (6) slides into the housing (4) and is securedusing rubber bands.

The relative height dimensions of the chamber (6) can vary relative tothe housing (4). Typically, the height dimension of the chamber (6) isgreater than the housing (4). Preferably, the height of the chamber (6)ranges from about 1.0 cm to about 7.0 cm, more preferably from about 2.0cm to about 6.0 cm, and even more preferably the from about 3.0 cm toabout 5.0 cm. The height of the housing (4) preferably ranges from about1.0 cm to about 3.0 cm, more preferably from about 1.5 cm to about 2.5cm, and even more preferably from about 1.8 cm to about 2.2 cm.

One embodiment of the present invention is a method to collect salivaproducts from ectoparasites using an apparatus of the present invention.Such a method is particularly advantageous because it enables isolationof ectoparasite saliva, including saliva proteins, substantially free ofcontaminating material. As such, the method can be used, for example, tocharacterize ectoparasite saliva proteins and to isolate ectoparasitesaliva proteins for diagnostic and therapeutic use.

One embodiment of the present method includes the steps of: (a)collecting ectoparasite saliva products on a collection means within asaliva collection apparatus which contains ectoparasites in the housingof the apparatus; and (b) extracting (i.e., eluting) the collectedectoparasite saliva products from the collection means with a solutionto form an extracted product-containing solution. Such an extractedsolution can be used directly as a formulation of the present inventionor can be submitted to further steps of fractionation and/orpurification as described in detail herein, to form additionalformulations of the present invention. Examples of such extractedsolutions include FS-1, FS-2 and FS-3.

In accordance with the present invention, a saliva collection apparatuscontaining the ectoparasites has an interface between the chamber andthe housing comprising a collection means capable of collecting at leasta portion of saliva products deposited by ectoparasite retained in theapparatus and a barrier means capable of substantially preventingcontaminating material from contacting the collection means. Theectoparasites contained in the apparatus are maintained under conditionssuch that there is a temperature differential between the chamber andthe housing; that is, the chamber of the apparatus has a temperaturewarmer than the temperature of the housing containing the ectoparasite,such that the warmer temperature in the chamber attracts theectoparasites retained in the housing to attempt to penetrate thebarrier means and collection means, thereby depositing saliva productson the collection means.

In one embodiment, the method of collecting saliva products includespre-wetting a collection means of the present invention prior topositioning the collection means in an apparatus of the presentinvention. A pre-wetting solution suitable for the present invention iscapable of facilitating the adsorption (i.e., collection) of salivaproducts in such a manner that the products can also be extracted duringan extraction step (i.e., when exposed to an appropriate solvent). Asuitable pre-wetting solution of the present invention includes anybuffer that is non-toxic to ectoparasites and has a physiological pH.Examples of suitable buffers include, phosphate buffered saline, water,phosphate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). A preferred prewetting solution includes sterilewater containing 50 U/ml penicillin and 50 μg/ml streptomycin.

When an apparatus used to collect saliva products includes a blottingmeans, that blotting means should be moistened either prior to orfollowing placement of the blotting means into the chamber. Preferredmoistening solutions include, but are not limited to, water, phosphatebuffered saline, phosphate buffer, tris buffer, HEPES buffer, TEA bufferand TES buffer. More preferred moistening solutions include water and 50U/ml penicillin and 50 μg/ml streptomycin. According to the presentinvention, a blotting means is sufficiently moistened to producehumidity in a chamber but not to drip liquid from which ectoparasitesretained in the apparatus can drink. In a preferred embodiment, ablotting means which is about 4.0 cm in diameter and about 2.5 mm thickis moistened with about 2.3 milliliters (ml) of moistening solution.

In one embodiment, a pre-determined number of ectoparasites areintroduced into the housing of an apparatus of the present invention.The number of ectoparasites to be introduced into a housing can varywith the size of the housing and should be a number that will not hinderthe ability of an ectoparasite to deposit saliva on a collection meansof the present invention.

In a preferred embodiment of the present invention, fleas are added toan apparatus of the present invention. Suitable and preferred numbers offleas to introduce into a housing are heretofore disclosed. Inparticular, fleas newly emerged from the pupal stage are used. Suchfleas can be raised as described in Wade et al., pp 186-190, 1988, J.Med Entomol., vol 25. Preferably, such fleas have not had a blood meal.Fleas can be loaded into an apparatus by placing the fleas in anaquarium and aspirating them into the housing under vacuum. Additionaloptional components suitable for the maintenance of fleas can be addedto the container, such as animal hair, and dry tissue.

In a preferred embodiment, at least one apparatus of the presentinvention having fleas contained in the housing of the apparatus isattached to an artificial feeding system such as disclosed herein. Theapparatus can remain attached to the feeding system as long as fleascontinue to release saliva while penetrating a collection means.Preferably, fleas are maintained in the apparatus attached to thefeeding system from about 12 hours to about 120 hours, more preferablyfrom about 24 hours to about 96 hours, and even more preferably forabout 72 hours since fleas essentially stop secreting saliva by aboutthat time. In accordance with the method of the present invention,preferably at least about 80 micrograms (μg), more preferably at leastabout 90 μg and even more preferably at least about 200 μg, of fleasaliva protein can be collected from about 10⁶ flea-hour when measuredusing a Bio-Rad Bradford assay (available from Bio-Rad, Hercules,Calif.).

According to the present invention, ectoparasite saliva products can beextracted using a solvent capable of extracting saliva products from acollection means of the present invention, preferably in a form suchthat the functional activities of the eluted products are maintained. Iffunctional activity of flea saliva proteins, for example is notmaintained, it is within the scope of the invention to re-fold proteinsto regain functionality using techniques known to those of skill in theart. Suitable extraction solvents include, but are not limited to,phosphate buffered saline, phosphate buffered saline containing sodiumchloride, TFA in acetonitrile, chaotropic agents, detergents, organics,salts or combinations thereof. Preferred extraction solvents includephosphate buffered saline, phosphate buffered saline containing sodiumchloride, acetonitrile and TFA in acetonitrile. More preferredextraction solvents include 1 M NaCl in phosphate buffered saline, 0.1%TFA in 50% acetonitrile, 1% TFA in 50% acetonitrile, 12.8% acetonitrileand 50% acetonitrile. Suitable extraction times for eluting proteins andother products from a collection means are described in detail in theExamples.

Further purifications of saliva proteins extracted from a collectionmeans of the present invention can be performed by fractionating theextracted product-containing solution to obtain separated peak fractionsand recovering at least one of the peak fractions substantially free ofthe remaining fractions to obtain a formulation of ectoparasite salivaproteins. In a preferred embodiment, proteins contained in extractedsaliva products of the present invention are further resolved bysubmitting the extract to HPLC purification to obtain peak fractions. Ina particularly preferred embodiment, extracted saliva proteins of thepresent invention are further resolved by HPLC to obtain the peakfractions shown in FIG. 2. Details regarding the extraction andresolution of such proteins are presented in the Examples.

According to the present invention, a formulation comprising at leastone ectoparasite saliva product of the present invention or a mimetopethereof, can be used to identify animals that are susceptible to or haveallergic dermatitis.

In accordance with the present invention, a “mimetope” refers to anycompound that is able to mimic the ability of an isolated ectoparasitesaliva product of the present invention to carry out its function (e.g.,anti-coagulation, anti-complement, vasodialators, proteases, acidphosphatases or detecting and/or treating the hypersensitivity of ananimal susceptible to or having allergic dermatitis). A mimetope can bea peptide that has been modified to decrease its susceptibility todegradation but that still retains the desired activity. Other examplesof mimetopes include, but are not limited to, carbohydrate-basedcompounds, lipid-based compounds, nucleic acid-based compounds, naturalorganic compounds, synthetically derived organic compounds,anti-idiotypic antibodies and/or catalytic antibodies, or fragmentsthereof. Mimetopes of the present invention can also includenon-proteinaceous portions of ectoparasite saliva products havingallergenic and/or antigenic activity (e.g., carbohydrate moietiesassociated with ectoparasite saliva proteins). A mimetope can beobtained by, for example, screening libraries of synthetic compounds forcompounds capable of altering the ability of ectoparasites to feed, orof detecting and/or treating allergic dermatitis resulting from thebites of ectoparasites. A mimetope can also be obtained by, for example,rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodelling. The predicted mimetope structures can then be produced by,for example, chemical synthesis, recombinant DNA technology, or byisolating a mimetope from a natural source (e.g., plants, animals,bacteria and fungi).

One embodiment of the present invention is an in vivo test that iscapable of detecting whether an animal is hypersensitive to ectoparasitesaliva products. An in vivo test of the present invention can initiallybe used to determine if an animal is hypersensitive to ectoparasitesaliva products and then used to determine if an animal ishypersensitive to a particular ectoparasite saliva component, inparticular to an ectoparasite saliva protein. An in vivohypersensitivity test of the present invention is particularly usefulfor identifying animals susceptible to or having allergic dermatitis. Anin vivo hypersensitivity test of the present invention is even moreuseful for identifying animals susceptible to or having FAD. A suitablein vivo hypersensitivity test of the present invention can be, but isnot limited to, a skin test comprising administering (e.g.,intradermally injecting or superficial scratching) an effective amountof a formulation containing at least one ectoparasite saliva product, ora mimetope thereof. Methods to conduct skin tests of the presentinvention are known to those of skill in the art and are brieflydisclosed herein.

Suitable formulations to use in an in vivo skin test includeectoparasite saliva components (i.e., saliva products collected on, andremaining absorbed to, a collection means of the present invention,ectoparasite saliva extracts, and one or more isolated ectoparasitesaliva proteins). A preferred formulation includes extracts and one ormore isolated proteins.

A suitable amount of ectoparasite saliva product for use in a skin testof the present invention can vary widely depending on the allergenicityof the product used in the test and on the site at which the product isdelivered. Suitable amounts of ectoparasite saliva products for use in askin test of the present invention include an amount capable of formingreaction, such as a detectable wheal or induration (hardness) resultingfrom an allergic reaction to the product. Preferred amounts ofectoparasite saliva extracts or proteins for use in a skin test of thepresent invention range from about 1 nanogram (ng) to about 500micrograms (μg), more preferably from about 5 ng to about 300 μg, andeven more preferably from about 10 ng to about 50 μg of ectoparasitesaliva extracts or proteins. It is to be appreciated by those of skillin the art that such amounts will vary depending upon the allergenicityof the extracts and/or protein(s) being administered.

According to the present invention, ectoparasite saliva products of thepresent invention can be combined with an immunopotentiator (e.g.,carriers or adjuvants of the present invention as defined in detailbelow). A novel aspect, however, of the present invention is that anectoparasite saliva product of the present invention can induce ahypersensitive response in the absence of an immunopotentiator.

A skin test of the present invention further comprises administering acontrol solution to an animal. A control solution can include a negativecontrol solution and/or a positive control solution. A positive controlsolution of the present invention contains an effective amount of atleast one compound known to induce a hypersensitive response whenadministered to an animal. A preferred compound for use as positivecontrol solution includes, but is not limited to, histamine. A negativecontrol solution of the present invention can comprise a solution thatis known not to induce a hypersensitive response when administered to ananimal. As such, a negative control solution can comprise a solutionhaving compounds essentially incapable of inducing a hypersensitiveresponse or simply a buffer used to prepare the formulation, such assaline. An example of a preferred negative control solution isphenolated phosphate buffered saline (available from Greer Laboratories,Inc., Lenoir, N.C.).

Hypersensitivity of an animal to one or more formulations of the presentinvention can be evaluated by measuring reactions (e.g., wheal size,induration or hardness; using techniques known to those skilled in theart) resulting from administration of one or more experimental sample(s)and control sample(s) into an animal and comparing the reactions to theexperimental sample(s) with reactions resulting from administration ofone or more control solution. Preferred devices for intradermalinjections include individual syringes. Preferred devices for scratchinginclude devices that permit the administration of a number of samples atone time. The hypersensitivity of an animal can be evaluated bydetermining if the reaction resulting from administration of aformulation of the present invention is larger than the reactionresulting from administration of a negative control, and/or bydetermining if the reaction resulting from administration of theformulation is at least about the same size as the reaction resultingfrom administration of a positive control solution. As such, if anexperimental sample produces a reaction greater than or equal to thesize of a wheal produced by administration of a positive control sampleto an animal, then that animal is hypersensitive to the experimentalsample. Conversely, if an experimental sample produces a reactionsimilar to the reaction produced by administration of a negative controlsample to an animal, then that animal is not hypersensitive to theexperimental sample.

Preferred wheal sizes for evaluation of the hypersensitivity of ananimal range from about 16 mm to about 8 mm, more preferably from about15 mm to about 9 mm, and even more preferably from about 14 mm to about10 mm in diameter.

Preferably, the ability or inability of an animal to exhibit animmediate hypersensitive response to a formulation of the presentinvention is determined by measuring wheal sizes from about 2 minutes toabout 30 minutes after administration of a sample, more preferably fromabout 10 minutes to about 25 minutes after administration of a sample,and even more preferably about 15 minutes after administration of asample.

Preferably, the ability or inability of an animal to exhibit a delayedhypersensitive response to a formulation of the present invention isdetermined by measuring induration and/or erythema from about 18 hoursto about 30 hours after administration of a sample, more preferably fromabout 20 hours to about 28 hours after administration of a sample, andeven more preferably at about 24 hours after administration of a sample.A delayed hypersensitivity response can also be measured using othertechniques such as by determining, using techniques known to those ofskill in the art, the extent of cell infiltrate at the site ofadministration during the time periods defined directly above.

In a preferred embodiment, a skin test of the present inventioncomprises intradermally injecting into an animal at a given site aneffective amount of a formulation that includes flea saliva extracts(i.e., flea saliva products extracted from a collection means of thepresent invention) or at least one flea saliva protein of the presentinvention, and intradermally injecting an effective amount of a controlsolution into the same animal at a different site. It is within thescope of one of skill in the art to use devices capable of deliveringmultiple samples simultaneously at a number of sites, preferablyenabling concurrent evaluation of numerous formulations. One preferredformulation comprises flea saliva products collected in accordance withthe present invention. Also preferred are formulations comprising one ormore recombinantly produced flea saliva proteins.

Suitable flea saliva products for use with a skin test of the presentinvention include FS-1, FS-2 and/or FS-3 as well as at least a portionof at least one flea saliva product that can be isolated from FS-1, FS-2and/or FS-3. A preferred flea saliva product for use with a skin testincludes FS-1, FS-2, FS-3 and/or at least a portion of one or more ofthe proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspf, fspG1,fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1,fspM2, fspN1, fspN2 and fspN3, or homologues thereof. A more preferredflea saliva product for use with a skin test includes FS-1, FS-2, FS-3and/or at least a portion of one or more of the proteins fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3. A yet more preferred flea salivaproduct for use with a skin test includes FS-1, FS-2, FS-3 and/or atleast a portion of one or more of the proteins fspG1, fspG2, fspG3,fspH, fspM1, fspM2, fspN1, fspN2 and fspN3. Such formulations are shownin the Examples section as being able to induce FAD in dogs. A preferredpositive control sample can be a sample comprising histamine. Apreferred negative control sample can be a sample comprising diluent.

Animals suitable and preferred to test for hypersensitivity toectoparasite saliva proteins using a skin test of the present inventionare disclosed herein. Particularly preferred animals to test with a skintest of the present invention include dogs, cats and horses, with dogsand cats being even more preferred.

Another embodiment of the present invention is an in vitroimmunoabsorbent test that is capable of detecting the presence of anantibody capable of binding to one or more ectoparasite saliva productsof the present invention by contacting a putative antibody-containingsolution with a solution containing ectoparasite saliva products in sucha manner that immunocomplexes can form and be detected. Thus, an invitro immunoabsorbent test of the present invention is particularlyuseful for identifying animals susceptible to or having allergicdermatitis by demonstrating that an animal has been previously exposedto an ectoparasite saliva antigen and, therefore may be hypersensitiveto further exposure to an ectoparasite saliva antigen.

According to the present invention, an in vitro hypersensitivity test ofthe present invention can be, but is not limited to, an immunoabsorbenttest comprising: (a) contacting a formulation of the present inventionwith a body fluid from an animal under conditions sufficient forformation of an immunocomplex between the formulation and antibodies, ifpresent, in the body fluid; and (b) determining the amount ofimmunocomplex formed, wherein formation of the immunocomplex indicatesthat the animal is susceptible to or has allergic dermatitis. Theimmunoabsorbent test is particularly useful for the detection of IgEantibodies in the body fluid, thereby indicating immediatehypersensitivity in the animal. Determining the amount of immunocomplexformed can include the step of separating depending on the mode ofdetection. Immunoabsorbent assays can be a variety of protocols and canbe set-up by those of skill in the art.

A preferred immunoabsorbent test of the present invention comprises afirst step of coating one or more portions of a solid substrate with asuitable amount of one or more ectoparasite saliva products of thepresent invention or a mimetope thereof, and of coating one or moreother portions of the (or another) solid substrate with a suitableamount of positive and/or negative control solutions of the presentinvention. A preferred solid substrate of the present invention caninclude, but is not limited to, an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, immunoblotmembranes and paper; a more preferred solid substrate includes an ELISAplate, a dipstick or a radioimmunoassay plate, with an ELISA plate and adipstick being even more preferred. As used herein, a dipstick refers toany solid material having a surface to which antibodies can be bound,such solid material having a stick-like shape capable if being insertedinto a test tube. Suitable and preferred flea saliva products for usewith an in vitro hypersensitivity test of the present invention are asdisclosed for a skin test of the present invention.

A second step of a preferred in vitro hypersensitivity test of thepresent invention comprises contacting the coated substrate with a bodyfluid, such as serum, plasma or whole blood, from an animal susceptibleto allergic dermatitis in such a manner as to allow antibodies containedin the body fluid that are capable of binding to ectoparasite salivaproducts to bind to such products bound to the substrate to formimmunocomplexes. Excess body fluid and antibodies are then washed fromthe substrate. In a preferred embodiment in which IgE antibodies in thebody fluid are to be measured, the body fluid can be pretreated toremove at least some of the other isotypes of immunoglobulin and/orother proteins, such as albumin, present in the fluid. Such removal caninclude, but is not limited to, contacting the body fluid with amaterial, such a Protein G, to remove IgG antibodies and/or affinitypurifying the IgE antibodies from other components of the body fluid byexposing the fluid to, for example, Concanavalin A (Con-A).

A third step of a preferred in vitro hypersensitivity test of thepresent invention comprises contacting the immunocomplexes bound to thesubstrate with a compound capable of binding to the immunocomplexes,such as a secondary antibody or other compound that is capable ofbinding to the heavy chain of allergy-related antibodies produced byanimals allergic to ectoparasites, in such a manner that the compound(s)can bind to the immunocomplexes. Preferred binding compounds include,but are not limited to, secondary antibodies capable of binding to theheavy chain of IgE antibodies. Preferred animals to test are disclosedherein. Compounds capable of binding to immunocomplexes are usuallytagged with a label which enables the amount of compound bound to theantibody from the body fluid to be measured. Such labels include, butare not limited to, a radioactive label, an enzyme capable of producinga color reaction upon contact with a substrate, a fluorescent label, achemiluminescent label, a chromophoric label or a compound capable ofbeing bound by another compound. Preferred labels include, but are notlimited to, fluorescein, radioisotopes, alkaline phosphatases, biotin,avidin, or peroxidases.

A fourth step of a preferred in vitro hypersensitivity test of thepresent invention comprises measuring the amount of detectable labelbound to the solid substrate using techniques known to those of skill inthe art. It is within the scope of the present invention that the amountof antibody from the body fluid bound to the substrate can be determinedusing one or more layers of secondary antibodies or other bindingcompounds. For example, an untagged secondary antibody can be bound to aserum antibody and the untagged secondary antibody can then be bound bya tagged tertiary antibody.

A hypersensitive animal is identified by comparing the level ofimmunocomplex formation using samples of body fluid with the level ofimmunocomplex formation using control samples. An immunocomplex refersto a complex comprising an antibody and its ligand (i.e., antigen). Assuch, immunocomplexes form using positive control samples and do notform using negative control samples. As such, if a body fluid sampleresults in immunocomplex formation greater than or equal toimmunocomplex formation using a positive control sample, then the animalfrom which the fluid was taken is hypersensitive to the ectoparasitesaliva product bound to the substrate. Conversely, if a body fluidsample results in immunocomplex formation similar to immunocomplexformation using a negative control sample, then the animal from whichthe fluid was taken is not hypersensitive to the ectoparasite salivaproduct bound to the substrate.

One embodiment of the present invention is a kit useful foridentification of an animal susceptible to or having allergicdermatitis. As used herein, a suspect animal is an animal to be tested.A kit of the present invention comprises a formulation of the presentinvention and a means for determining if an animal is susceptible to orhas allergic dermatitis, in which the formulation is used to identifyanimals susceptible to or having allergic dermatitis. A means fordetermining if an animal is susceptible to or has allergic dermatitiscan include an in vivo or in vitro hypersensitivity test of the presentinvention as described in detail above. A kit of the present inventionfurther comprises at least one control solution such as those disclosedherein.

A preferred kit of the present invention comprises the elements usefulfor performing an immunoassay. A kit of the present invention cancomprise one or more experimental samples (i.e., formulations of thepresent invention) and one or more control samples bound to at least onepre-packed dipstick, and the necessary means for detecting immunocomplexformation (e.g., labelled secondary antibodies or other bindingcompounds and any necessary solutions needed to resolve such labels, asdescribed in detail above) between antibodies contained in the bodilyfluid of the animal being tested and the proteins bound to the dipstick.It is within the scope of the invention that the kit can comprise simplya formulation of the present invention and that the detecting means canbe provided in another way.

An alternative preferred kit of the present invention comprises elementsuseful for performing a skin test. A kit of the present invention cancomprise at least one pre-packed syringe and needle apparatus containingone or more experimental samples and/or one or more control samples.

It is within the scope of the present invention that two or moredifferent in vivo and/or in vitro tests can be used in combination fordiagnostic purposes. For example, the immediate hypersensitivity of ananimal to an ectoparasite saliva allergen can be tested using an invitro immunoabsorbent test capable of detecting IgE antibodies specificfor an ectoparasite saliva allergen in the animal's bodily fluid. Whilemost animals that display delayed hypersensitivity to an ectoparasitesaliva allergen also display immediate hypersensitivity to the allergen,a small number of animals that display delayed hypersensitivity to anallergen do not display immediate hypersensitivity to the allergen. Insuch cases, following negative results from the IgE-specific in vitrotest, the delayed hypersensitivity of the animal to an ectoparasitesaliva allergen can be tested using an in vivo test of the presentinvention.

Another aspect of the present invention includes treating animalssusceptible to or having allergic dermatitis, with a formulation of thepresent invention. According to the present invention, the termtreatment can refer to the regulation of a hypersensitive response by ananimal to bites from ectoparasites. Regulation can include, for example,immunomodulation of cells involved in the animal's hypersensitiveresponse or alteration of the ability of an ectoparasite to introduceallergens into an animal, for example by inhibiting the anti-coagulationactivity of a saliva enzyme, thereby impairing the ability of thearthropod to penetrate the dermis of an animal and feed.Immunomodulation can include modulating the activity of moleculestypically involved in an immune response (e.g., antibodies, antigens,major histocompatibility molecules (MHC) and molecules co-reactive withMHC molecules). In particular, immunomodulation refers to modulation ofantigen:antibody interactions resulting in inflammatory responses,immunosuppression, and immunotolerization of cells involved in ahypersensitive response. Immunosuppression refers to inhibiting animmune response by, for example, killing particular cells involved inthe immune response. Immunotolerization refers to inhibiting an immuneresponse by energizing (i.e., diminishing reactivity of a T cell to anantigen) particular cells involved in the immune response. Suitable andpreferred ectoparasites against which to treat an animal are disclosedherein. A particularly preferred formulation of the present invention isused to treat FAD.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is usefulfor immunomodulating the immune response of the animal (i.e.,immunomodulating the animal) so as to block (i.e., to inhibit, reduce orsubstantially prevent) a hypersensitive response by the animal uponsubsequent exposure to allergenic components transmitted through bitesfrom ectoparasites. Such a therapeutic composition is useful forimmunomodulating animals known to be hypersensitive to ectoparasitesaliva products and animals susceptible to hypersensitive responsesagainst ectoparasite saliva products.

One embodiment of the present invention is a therapeutic compositionthat includes de-sensitizing compounds capable of inhibiting an immuneresponse to an ectoparasite saliva product of the present invention.Such de-sensitizing compounds include blocking compounds, toleragensand/or suppressor compounds. Blocking compounds comprise compoundscapable of modulating antigen:antibody interactions that can result ininflammatory responses, toleragens are compounds capable ofimmunotolerizing an animal, and suppressor compounds are capable ofimmunosuppressing an animal. A de-sensitizing compound of the presentinvention can be soluble or membrane-bound. Membrane-boundde-sensitizing compounds can be associated with biomembranes, includingcells, liposomes, planar membranes or micelles. A soluble de-sensitizingcompound of the present invention is useful for: (1) inhibiting a Type Ihypersensitivity reaction by blocking IgE:antigen mediatedde-granulation of mast cells; (2) inhibiting a Type III hypersensitivityreaction by blocking IgG:antigen complex formation leading to complementdestruction of cells; and (3) inhibiting a Type IV hypersensitivityreaction by blocking T helper cell stimulation of cytokine secretion bymacrophages. A membrane-bound de-sensitizing compound of the presentinvention is useful for: (1) inhibiting a Type II hypersensitivityreaction by blocking IgG:antigen complex formation on the surface ofcells leading to complement destruction of cells; (2) inhibiting a TypeII hypersensitivity reaction by blocking IgG regulated signaltransduction in immune cells; and (3) inhibiting a Type IVhypersensitivity reaction by blocking T cytotoxic cell killing ofantigen-bearing cells.

A de-sensitizing compound of the present invention can also becovalently linked to a ligand molecule capable of targeting thede-sensitizing compound to a specific cell involved in a hypersensitiveresponse to ectoparasite saliva products. Appropriate ligands with whichto link a de-sensitizing compound include, for example, at least aportion of an immunoglobulin molecule, cytokines, lectins, heterologousallergens, CD8 molecules or major histocompatibility molecules (e.g.,MHC class I or MHC class II molecules). Preferred portions ofimmunoglobulin molecules to link to a de-sensitizing compound includevariable regions capable of binding to immune cell specific surfacemolecules and constant regions capable of binding to Fc receptors onimmune cells, in particular IgE constant regions. Preferred CD8molecules include at least the extracellular functional domains of the αchain of CD8. An immune cell refers to a cell involved in an immuneresponse, in particular, cells having MHC class I or MHC class IImolecules. Preferred immune cells include antigen presenting cells, Tcells and B cells.

In one embodiment, a therapeutic composition of the present inventionincludes ectoparasite saliva products of the present invention, ormimetopes thereof. Preferred therapeutic compositions includeformulations comprising ectoparasite saliva extracts or at least oneectoparasite saliva product (preferably protein) of the presentinvention or mimetopes thereof.

Suitable therapeutic compositions of the present invention for treatingflea allergy dermatitis include flea saliva extracts and otherformulations including at least one flea saliva product, preferably aprotein, or a mimetope thereof. Preferred therapeutic compositionsinclude FS-1, FS-2 and/or FS-3 as well as at least a portion of at leastone flea saliva product that can be isolated from FS-1, FS-2 and/orFS-3. As such, preferred formulations for use as therapeuticcompositions include FS-1, FS-2, FS-3, and/or at least a portion of oneor more of the proteins fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE,fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2,fspM1, fspM2, fspN1, fspN2 and fspN3, or homologues thereof. A morepreferred flea saliva extract for use as a therapeutic compositionsincludes FS-1, FS-2, FS-3, and/or at least a portion of one or more ofthe proteins fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2,fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3. A yet morepreferred flea saliva extract for use as a therapeutic compositionsincludes FS-1, FS-2, and/or at least a portion of one or more of theproteins fspG1, fspG2, fspG3, fspH, fspM1, fspM2, fspN1, fspN2 andfspN3.

In another embodiment, a therapeutic composition can includeectoparasite products of the present invention associated with asuitable excipient. A therapeutic composition of the present inventioncan be formulated in an excipient that the animal to be treated cantolerate. Preferred excipients are capable of maintaining a product ofthe present invention in a form that is capable of being bound by cellsinvolved in an allergic response in an animal such that the cells arestimulated to initiate or enhance an immune response. Examples of suchexcipients include water, saline, Ringer's solution, dextrose solution,Hank's solution, and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyloleate, or triglycerides may also be used. Other useful formulationsinclude suspensions containing viscosity enhancing agents, such assodium carboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In another embodiment, a therapeutic composition of the presentinvention can also comprise a carrier or adjuvant, although it is to beappreciated that an advantage of saliva products of the presentinvention is that adjuvants and/or carriers are not required foradministration. Adjuvants are typically substances that generallyenhance the immune response of an animal to a specific antigen. Suitableadjuvants include, but are not limited to, Freund's adjuvant; otherbacterial cell wall components; aluminum-based salts; calcium-basedsalts; silica; polynucleotides; toxoids; serum proteins; viral coatproteins; other bacterial-derived preparations; gamma interferon; blockcopolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc.Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research,Inc., Hamilton, Mont.); and saponins and their derivatives, such as QuilA (available from Superfos Biosector A/S, Denmark).

Carriers are typically compounds that increase the half-life of atherapeutic composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled releaseformulations, biodegradable implants, liposomes, bacteria, viruses,oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a therapeuticcomposition of the present invention into the bloodstream of an animal.Suitable controlled release formulations include, but are not limitedto, biocompatible (including biodegradable) polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ.

The present invention also includes a recombinant virus particletherapeutic composition. Such a composition includes a recombinantmolecule of the present invention that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient. A number of recombinantvirus particles can be used, including, but not limited to, those basedon alphaviruses, poxviruses, adenoviruses, herpesviruses, andretroviruses. Preferred recombinant particle viruses are those based onalphaviruses (such as Sindbis virus), herpesviruses and poxviruses.Methods to produce and use recombinant virus particle vaccines aredisclosed in U.S. patent application Ser. No. 08/015/414, filed Feb. 8,1993, entitled “Recombinant Virus Particle Vaccines”, which isincorporated by reference herein in its entirety.

When administered to an animal, a recombinant virus particle therapeuticcomposition of the present invention infects cells within the immunizedanimal and directs the production of a protective protein or RNA nucleicacid molecule that is capable of protecting the animal from allergicdermatitis caused by the bites of ectoparasites. For example, arecombinant virus particle comprising a nucleic acid molecule encodingone or more ectoparasite saliva protein of the present invention isadministered according to a protocol that results in the tolerization ofan animal against ectoparasite saliva allergens.

Therapeutic compositions of the present invention can be sterilized byconventional methods which do not result in protein degradation (e.g.,filtration) and/or lyophilized.

A therapeutic composition of the present invention can be administeredto any animal susceptible to ectoparasite infestation as hereindescribed. Acceptable protocols by which to administer therapeuticcompositions of the present invention in an effective manner can varyaccording to individual dose size, number of doses, frequency of doseadministration, and mode of administration. Determination of suchprotocols can be accomplished by those skilled in the art. An effectivedose refers to a dose capable of treating an animal againsthypersensitivity to ectoparasite saliva allergens. Effective doses canvary depending upon, for example, the therapeutic composition used, thearthropod from which the composition was derived, and the size and typeof the recipient animal. Effective doses to immunomodulate an animalagainst ectoparasite saliva allergens include doses administered overtime that are capable of alleviating a hypersensitive response by ananimal to ectoparasite saliva allergens. For example, a first tolerizingdose can comprise an amount of a therapeutic composition of the presentinvention that causes a minimal hypersensitive response whenadministered to a hypersensitive animal. A second tolerizing dose cancomprise a greater amount of the same therapeutic composition than thefirst dose. Effective tolerizing doses can comprise increasingconcentrations of the therapeutic composition necessary to tolerize ananimal such that the animal does not have a hypersensitive response tothe bite of an ectoparasite. An effective dose to desensitize an animalcan comprise a concentration of a therapeutic composition of the presentinvention sufficient to block an animal from having a hypersensitiveresponse to the bite of an ectoparasite. Effective desensitizing dosescan include repeated doses having concentrations of a therapeuticcomposition that cause a minimal hypersensitive response whenadministered to a hypersensitive animal.

A suitable single dose is a dose that is capable of treating an animalagainst hypersensitivity to ectoparasite saliva allergens whenadministered one or more times over a suitable time period. For example,a preferred single dose of an ectoparasite saliva product, or mimetopetherapeutic composition is from about 0.5 ng to about 1 g of thetherapeutic composition per kilogram body weight of the animal. Furthertreatments with the therapeutic composition can be administered fromabout 1 hour to 1 year after the original administration. Furthertreatments with the therapeutic composition preferably are administeredwhen the animal is no longer protected from hypersensitive responses toectoparasite. Particular administration doses and schedules can bedeveloped by one of skill in the art based upon the parameters discussedabove. Modes of administration can include, but are not limited to,subcutaneous, intradermal, intravenous, nasal, oral, transdermal andintramuscular routes.

A therapeutic composition of the present invention can be used inconjunction with other compounds capable of modifying an animal'shypersensitivity to ectoparasite bites. For example, an animal can betreated with compounds capable of modifying the function of a cellinvolved in a hypersensitive response, compounds that reduce allergicreactions, such as by systemic agents or anti-inflammatory agents (e.g.,anti-histamines, anti-steroid reagents, anti-inflammatory reagents andreagents that drive immunoglobulin heavy chain class switching from IgEto IgG). Suitable compounds useful for modifying the function of a cellinvolved in a hypersensitive response include, but are not limited to,antihistamines, cromolyn sodium, theophylline, cyclosporin A, adrenalin,cortisone, compounds capable of regulating cellular signal transduction,compounds capable of regulating adenosine 3′, 5′-cyclic phosphate (cAMP)activity, and compounds that block IgE activity, such as peptides fromIgE or IgE specific Fc receptors, antibodies specific for peptides fromIgE or IgE-specific Fc receptors, or antibodies capable of blockingbinding of IgE to Fc receptors.

Another aspect of the present invention includes a method forprescribing treatment for animals susceptible to or having allergicdermatitis, using a formulation of the present invention. A preferredmethod for prescribing treatment for flea allergy dermatitis, forexample, comprises: (1) intradermally injecting into an animal at onesite an effective amount of a formulation containing at least one fleasaliva antigen of the present invention, or a mimetope thereof (suitableand preferred formulations are disclosed herein); (2) intradermallyinjecting into the animal at a second site an effective amount of acontrol solution; (3) evaluating if the animal has flea allergydermatitis by measuring and comparing the wheal size resulting frominjection of the formulation with the wheal size resulting frominjection of the control solution; and (4) prescribing a treatment forthe flea allergy dermatitis.

An alternative preferred method for prescribing treatment for fleaallergy dermatitis comprises: (1) contacting a first portion of a sampleof bodily fluid obtained from an animal to be tested with an effectiveamount of a formulation containing at least one flea saliva antigen, ora mimetope thereof (suitable and preferred formulations are disclosedherein) to form a first immunocomplex solution; (2) contacting apositive control antibody to form a second immunocomplex solution; (3)evaluating if the animal has flea allergy dermatitis by measuring andcomparing the amount of immunocomplex formation in the first and secondimmunocomplex solutions; and (4) prescribing a treatment for the fleaallergy dermatitis. It is to be noted that similar methods can be usedto prescribe treatment for allergies caused by other ectoparasites usingectoparasite saliva product formulations as disclosed herein.

Another aspect of the present invention includes a method for monitoringanimals susceptible to or having allergic dermatitis, using aformulation of the present invention. In vivo and in vitro tests of thepresent invention can be used to test animals for allergic dermatitisprior to and following any treatment for allergic dermatitis. Apreferred method to monitor treatment of flea allergy dermatitis (whichcan also be adapted to monitor treatment of other ectoparasiteallergies) comprises: (1) intradermally injecting an animal at one sitewith an effective amount of a formulation containing at least one fleasaliva product, or a mimetope thereof (suitable and preferredformulations are disclosed herein); (2) intradermally injecting aneffective amount of a control solution into the animal at a second site;and (3) determining if the animal is desensitized to flea salivaantigens by measuring and comparing the wheal size resulting frominjection of the formulation with the wheal size resulting frominjection of the control solution.

An alternative preferred method to monitor treatment of flea allergydermatitis (which can be adapted to monitor treatments of otherectoparasite allergies) comprises: (1) contacting a first portion of asample of bodily fluid obtained from an animal to be tested with aneffective amount of a formulation containing at least one flea salivaproduct or mimetope thereof (suitable and preferred formulations aredisclosed herein) to form a first immunocomplex solution; (2) contactinga positive control antibody to form a second immunocomplex solution; and(3) determining if the animal is desensitized to flea saliva antigens bymeasuring and comparing the amount of immunocomplex formation in thefirst and second immunocomplex solutions.

The present invention also includes antibodies capable of selectivelybinding to an ectoparasite saliva product, or mimetope thereof. Such anantibody is herein referred to as an anti-ectoparasite saliva productantibody. As used herein, the term “selectively binds to” refers to theability of such an antibody to preferentially bind to ectoparasitesaliva products and mimetopes thereof. In particular, the presentinvention includes antibodies capable of selectively binding to fleasaliva products. Binding can be measured using a variety of methodsknown to those skilled in the art including immunoblot assays,immunoprecipitation assays, enzyme immunoassays (e.g., ELISA),radioimmunoassays, immunofluorescent antibody assays and immunoelectronmicroscopy; see, for example, Sambrook et al., ibid.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein or mimetope used to obtain the antibodies. Preferredantibodies are raised in response to ectoparasite saliva proteins, ormimetopes thereof. More preferred antibodies are raised in response toat least one ectoparasite saliva protein, or mimetope thereof, having atleast a portion of an ectoparasite saliva protein eluted from acollection means of the present invention. Even more preferredantibodies are raised in response to at least one flea saliva product,or homologues thereof (e.g., saliva products of other ectoparasites),contained in the saliva extracts FS-1, FS-2 and/or FS-3. More preferredectoparasite saliva proteins against which to raise an antibody includesat least a portion of one or more of the proteins fspA, fspB, fspC1,fspC2, fspD1, fspD2, fspE, fspf, fspG1, fspG2, fspG3, fspH, fspI, fspJ1,fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3, orhomologues thereof. Preferably, an antibody of the present invention hasa single site binding affinity of from about 10³ M⁻¹ to about 10¹² M⁻¹for a flea saliva product of the present invention.

A preferred method to produce antibodies of the present inventionincludes administering to an animal an effective amount of anectoparasite saliva product or mimetope thereof to produce the antibodyand recovering the antibodies. Antibodies raised against definedproducts or mimetopes can be advantageous because such antibodies arenot substantially contaminated with antibodies against other substancesthat might otherwise cause interference in a diagnostic assay or sideeffects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as vaccines to passively immunize an animalin order to protect the animal from allergic dermatitis, (b) as positivecontrols in test kits, and/or (c) as tools to recover desiredectoparasite saliva products from a mixture of proteins and othercontaminants.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This example describes the collection of flea saliva proteins using asaliva collection apparatus of the present invention.

A saliva collection apparatus was prepared as follows. Referring toFIGS. 4A and 4B, a humidifying means (34) comprising about 4 pieces ofVWR blotting pads #320 (VWR, Denver, Colo.) was prepared that fit theinner diameter (about 47 mm in diameter) of a chamber (6) of a salivacollection apparatus (2). The blotting pads were pre-wetted using asufficient amount of pre-wetting solution (sterile water containing 50units/ml penicillin and 50 μg/ml streptomycin, available from Sigma, St.Louis, Mo.) such that the blotting pads were damp but not dripping wet.The pre-wetted filters (34) were placed inside the bottom end (22) ofthe chamber (6) of the saliva collection apparatus (2) such that thefilter paper sat immediately inside the bottom end (22) of the chamber(6).

A collection means (24) comprising a Durapore™ membrane (available fromMillipore, Bedford, Mass.) was cut to fit the outer diameter (about 48mm in diameter) of the chamber (6) of the saliva collection apparatus(2). The Durapore™ membrane was pre-wetted using the pre-wettingsolution described above. The Durapore™ membrane (24) was placedimmediately outside the bottom end (22) of the chamber (6) such that theDurapore™ membrane (24) contacted the outer rim of the bottom end (22)of the chamber (6) and also contacted the damp filter paper. A barriermeans comprising a piece of stretched Parafilm™ (28) (available fromAmerican National Can™, Greenwich, Conn.) was stretched over thecollection means (24) and bottom end (22) of the chamber (6) and up theouter wall (30) of the chamber (6). A rubber seal (32) (i.e., an O-ring)was placed over the Parafilm™ (28) thereby further securing theParafilm™ (28) across the collection means (24) and to the outer wall(30) and to seal in the chamber (6) environment.

The collection apparatus (2) was preassembled and then the top end (20)of the chamber (6) was attached to an artificial feeding system capableof acting as a source of heat and humidity such as that described byWade et al., (ibid.). The artificial feeding system comprised a largeplexiglass box (40 cm×40 cm×40 cm) divided horizontally into an uppercompartment and a lower compartment by a plexiglass shelf having holesdrilled through. A collection apparatus (2) was inserted into a holesuch that the chamber (6) of the apparatus (2) was located above theshelf in the upper compartment and the housing (4) was located below theshelf in the lower compartment. The apparatus (2) was secured to theshelf by attaching a rubber band attached to metal hooks placed in theshelf. Any open holes in the shelf were closed off using rubber stoppersto isolate the environment within the upper compartment from theenvironment within the lower compartment. The upper compartmentcontained two trays of water, a fan and a heating block. The trays ofwater were placed such that the fan faced the trays. While the apparatus(2) was maintained in the artificial feeding system, the fan was blowncontinuously thereby circulating heat and humidity throughout the uppercompartment and the chamber (6) of the collection apparatus (2). Assuch, the relative humidity within the chamber (6) was maintained atabout 94% humidity and the temperature was maintained at about 37° C.

About 3,000 to 5,000 newly emerged unfed Ctenocephalides felis fleaswere added to the housing (4) of the collection apparatus (2). The fleaswere first collected in a 20 gallon glass aquarium. The fleas were thentransferred to the housing (4) of a collection apparatus (2) by placingthe end of the housing (4) having the nylon mesh of the exchange means(16) on top of a vacuum chamber and aspirating the fleas from theaquarium into the housing (4) through a tygon tubing. The housing (4)was then covered with the nylon mesh of the retaining means (18) tosecure the fleas within the housing (4). The bottom end (22) of thechamber (6) was then placed on the housing (4) such that the Parafilm™(28) and the nylon mesh of the retaining means (18) were juxtaposed.When the collection apparatus (2) was attached to the artificial feedingsystem, the ambient temperature within the housing (4) was maintained atabout 27° C. while the ambient temperature of the chamber (6) wasmaintained at about 37° C. The relative humidity of the housing (4) wasmaintained at about 50% by closing the lower compartment with theplexiglass shelving.

In one experiment, flea saliva products were collected on a Durapore™membrane (24) and visualized by immersing the membrane in 0.1% Coomassieblue stain for 20 minutes, destaining the membrane in 50% methanol andair drying the membrane. Proteins deposited on the membrane weredetected by their blue color.

In another experiment, flea saliva products were collected for 0 through24 hours, 24 through 72 hours, and 72 through 120 hours after loadingfleas into the collection apparatus. At 24 hours, 72 hours and 120hours, the Durapore™ membrane (24) attached to the collection apparatus(2) was removed and a new pre-wetted Durapore™ membrane (24) wasattached to the same apparatus. The blotting pads were re-wetted usingthe pre-wetting solution described above when the new Durapore™ membrane(24) was replaced. Flea saliva products were extracted from theDurapore™ membrane (24) by soaking each membrane from each time pointseparately in a solvent comprising 50% acetonitrile with 1% TFAovernight at room temperature with stirring to obtain a flea salivaproduct mixture comprising flea saliva products that had eluted into thesolvent. The mixture containing the flea saliva products was recoveredand lyophilized until dry to form a pellet. The amount andcharacteristics of flea saliva proteins eluted from each Durapore™membrane from each time point was determined by reducing 14%Tris-glycine SDS-PAGE using techniques similar to those described bySambrook et al., ibid. The resultant protein pattern was visualized bystaining the gel with Coomassie blue stain using techniques as describedabove. The amount of saliva proteins collected on the membranesdecreased when the fleas had been in the collection apparatus for morethan 72 hours.

Example 2

Standard procedures to collect FS-1, FS-2 and FS-3 flea saliva extractsof the present invention were performed as follows. Flea saliva productswere collected for 72 hours on collection membranes using the methoddescribed in Example 1, except that for flea saliva extract FS-3, thecollection membrane was DE-81 chromatography paper, available fromWhatman, Inc., Clifton, N.J.

A. Flea Saliva Extracts FS-1 and FS-2:

Flea saliva products were extracted from the Durapore™ membrane (24) bysoaking each membrane from each time point separately in a first solventcomprising 50% acetonitrile with 0.1% TFA for 8 hours. The first mixturecontaining the eluted flea saliva products was recovered and lyophilizeduntil dry, thereby forming a first pellet. The same membranes were thensoaked in a second solvent comprising 50% acetonitrile with 1% TFAovernight at room temperature with stirring to obtain a flea salivaproduct mixture comprising flea saliva products that had eluted into thesecond solvent. The second mixture was recovered from this secondextraction and lyophilized until dry to form a second pellet.

The two pellets recovered from the two lyophilization steps were mixedwith a third solvent comprising 12.8% acetonitrile and flea salivaproducts solubilized in the solvent were recovered. The non-solubilizedmaterial was mixed again with 12.8% acetonitrile and additional fleasaliva products solubilized in the solvent were recovered. The twomixtures were combined to obtain the extract FS-1.

The non-solubilized material remaining after the second solubilizationstep was then mixed with 50% acetonitrile which solubilized theremaining material to obtain the extract FS-2.

The amount and characteristics of flea saliva proteins contained in theFS-1 and FS-2 flea saliva extracts obtained in at least one experimentwere determined according to the following method. Each extract wasconcentrated by evaporation under vacuum and evaluated by reducing 16%Tris-glycine SDS-PAGE using techniques similar to those described bySambrook et al., ibid. Using such standard procedures, about 10 μg ofFS-1 or FS-2 eluted from the Durapore™ membrane was loaded onto a 16%Tris-glycine polyacrylamide gel and subjected to electrophoresis underreducing conditions. The gel was stained with Coomassie blue and dried.

The results are shown in FIG. 1B. FS-1 is shown in lane 13 of FIG. 1Band FS-2 is shown in lanes 14 and 15 of FIG. 1B. FS-1 was found tocontain proteins estimated to have the following molecular weights: 9kD, 11 kD, 12 kD, 15 kD, 22 kD, 48 kD, 50 kD, 53 kD, 80 kD, 124 kD, 130kD, 189 kD and 201 kD. Those proteins of 80 kD and above were muchfainter than the lower molecular weight bands. FS-2 was found to containproteins having the following molecular weights: 47 kD, 49 kD, 52 kD, 57kD, 64 kD, 71 kD, 88 kD, 96 kD, 97 kD, 130 kD, 161 kD, 175 kD, 189 kD,222 kD, 235 kD and 302 kD. The bands at 47 kD, 49 kD and 52 kD were moreprominent than the bands having higher molecular weights. The resultssuggest that a substantial portion of the protein contained in FS-2 isfspN1, fspN2 and/or fspN3.

Protein concentrations were measured using a Bio-Rad Bradford assay(available from Bio-Rad, Hercules, Calif.). The results indicate thatabout 750 μg of protein can be collected in about 3.66×10⁷ flea hours(5.08×10⁵ fleas for 72 hours) in an FS-1 extract and about 2.35 mg ofprotein can be collected in about 3.66×10⁷ flea hours in an FS-2extract.

B. Flea Saliva Extract FS-3:

Flea saliva products to produce FS-3 flea saliva extract were collectedin a manner similar to the method by which FS-1 and FS-2 were collected,except that the collection membrane (24) was DE-81 chromatography paper.Flea saliva products were extracted from the DE-81 membrane by soakingeach membrane from each time point separately in a solvent comprising 1MNaCl in phosphate buffered saline for about 8 hours. The products wererecovered from the solvent using standard techniques, such as disclosedfor FS-1 and FS-2.

Analysis of an FS-3 flea saliva extract indicated that FS-3 appeared tocontain proteins such as those found in FS-1 and FS-2, at least based on1-dimensional gel electrophoresis. The SDS-PAGE pattern of FS-3, forexample, was very similar to that of FS-1 except that there appeared tobe increased quantities of the higher molecular weight proteins in theFS-3 extract. FS-3 flea saliva extract was also shown to haveanti-coagulation activity, using techniques standard in the art; see,for example, Dunwiddle et al., 1991, Thrombosis Research 64, 787-794;Ribeiro et al., 1990, Comp. Biochem. Physiol. 95, 215-218; Ribeiro etal., 1990, Br. J. Pharmacol. 101, 932-936; Ribeiro et al., 1987, Exper.Parasitol. 64, 347-353; Cupp et al., 1994, Am. J. Trop. Med. Hyg. 50,241-246; Garcia et al., 1994, Exper. Parasitol. 78, 287-293. The FS-3extract was also shown to exhibit acid phosphatase activity, usingtechniques standard in the art, such as those supplied by Sigma, St.Louis, Mo., with the Sigma acid phosphtase assay kit.

Example 3

This example describes the characterization by HPLC of flea salivaproteins collected using a saliva collection apparatus of the presentinvention.

An FS-1 flea saliva extract was collected as described in Example 2 fromabout 140,000 fleas for 72 hours. Proteins contained in FS-1 wereresolved using standard procedures of high pressure liquidchromatography (HPLC). Specifically, the proteins were passed over a 15cm×0.46 cm C4 column using a gradient from 0.1% TFA in water (Solvent A)to 0.085% TFA in 90% CH₃CN (Solvent B) at a flow rate of 0.8 ml perminute. Thus, the gradient was about 5.6% Solvent B at 15 minutes andabout 100% Solvent B at 75 minutes.

The results are shown in FIG. 2. About 14 major protein fractions wereresolved. The recovery for each peak was about 5 μg to 10 μg of proteinper peak. The peaks were labelled peak A, peak B, peak C, peak D, peakE, peak F, peak G, peak H, peak I, peak J, peak K, peak L, peak M andpeak N, as shown in FIG. 2, and represent, respectively, proteinformulations fspA, fspB, fspC1 and fspC2, fspD1 and fspD2, fspE, fspF,fspG1, fspG2, fspG3, fspH, fspI, fspJ1 and fspJ2, fspK, fspL1 and fspL2,fspM1 and fspM2, and fspN1, fspN2 and fspN3.

Samples from each HPLC peak were resolved by Tris Glycine SDS-PAGE gelsusing the method described in Example 1. The results are shown in FIGS.1A, 1B and 1C. The proteins shown in FIGS. 1A and 1B were resolved on16% Tris Glycine SDS-PAGE gels and the proteins shown in FIG. 1C wereresolved on a 14% Tris Glycine SDS-PAGE gel. Protein markers are shownin lane 1 of FIG. 1A, lane 2 of FIG. 1B and lane 1 of FIG. 1C. Theadditional lanes show saliva formulation samples as follows:

FIG. 1A Lane Fraction(s) Fs-( ) 1) — Mol. Wt. Std. 2) 10 — 3) 11-13 A 4)14 B 5) 15 B 6) 16 C1 7) 17 C2 8) 18 D1 9) 19 D1 10)  20 D2 11)  21 D212)  22 E 13)  23 F 14)  24 G 15)  25 G

Fig. 1B Lane Fraction(s) Fs-( ) 1) 26-27 G 2) — Mol. Wt. Std. 3) 28 H 4)29-30 I 5) 31 J 6) 32 K 7) 33 K 8) 34 L 9) 35 M1 10)  36-37 M1 11)  38M1 12)  39-50 M2 13)  — FS-1 14)  — FS-2 15)  — FS-2

FIG. 1C Lane Fraction(s) FS-( ) 1) — Mol. Wt. Std. 2) 56-68 N

Referring to FIG. 1A, the following flea saliva proteins (referred to asbands) were observed: a prominent band of about 10 kD in peak A and peakB samples; a prominent band of about 6 kD and a less prominent band of 9kD in a peak C sample referred to as C1; a prominent band of about 7 kDin a peak C sample referred to as C2; a prominent band of about 7 kD anda less prominent band of 8 kD in a peak D sample referred to as D1; aprominent band of about 8 kD and a less prominent band of 9 kD in a peakD sample referred to as D2; a prominent band of 8 kD and a lessprominent band of about 7 kD in peaks E and F samples; and a prominentband of about 9 kD, and less prominent bands of about 7 kD and 10 kD ina peak G sample. Referring to FIG. 1B, the following flea salivaproteins were observed: a prominent band of about 9 kD and a lessprominent band of about 12 kD in a peak H sample; a prominent band ofabout 21 kD, and less prominent bands of about 7 kD, 9 kD, 12 kD, 14 kD,and 70 kD in a peak I sample; prominent bands of about 14 kD and 21 kD,and less prominent bands of about 11 kD and 17 kD in a peak J sample;prominent bands of about 14 kD and 15 kD and less prominent bands ofabout 12 kD, 18 kD and 21 kD in a peak K sample; a prominent band ofabout 15 kD in a peak L sample; prominent bands of about 11 kD, 12 kDand 21 kD and less prominent bands of about 15 kD, 17 kD, 22 kD and 37kD in a peak M sample referred to as M1; and a prominent band of about36 kD and less prominent bands of about 11 kD, 21 kD and 22 kD in a peakM sample referred to as M2. Referring to FIG. 1C, prominent bands ofabout 42 kD, 43 kD and 44 kD and a less prominent band of about 32 kDwere detected in a peak N sample.

Example 4

This example describes the amino acid sequence analysis of the isolatedand HPLC purified flea saliva proteins.

Amino (N-) terminal amino acid sequencing analysis was performed onseveral of the HPLC-separated flea saliva proteins described in Example3 using standard procedures known to those in the art (see, for example,Geisow et al., 1989, in Protein Sequencing: A Practical Approach, J B CFindlay and M J Geisow (eds.), IRL Press, Oxford, England, pp. 85-98;Hewick et al., 1981, J. Biol. Chem., Vol. 256, pp. 7990-7997).

The N-terminal partial amino acid sequence of flea saliva protein fspA,which migrated as Peak A in FIG. 2, was determined to be

Y G K Q Y S E K G G R G Q R H Q I L K K G K Q YS           S K       I     L   D   L S Ras represented in standard single letter code. This N-terminal partialamino acid sequence of fspA is denoted SEQ ID NO:1. It should be notedthat there was heterogeneity in several positions which may representsequence errors (i.e., misidentification of amino acids) or allelicvariations in the flea population from which the saliva proteins werecollected. There was an apparently equal likelihood of finding any oneof the alternative amino acids at the indicated positions.

The N-terminal partial amino acid sequence of flea saliva protein fspB,which migrated as Peak B in FIG. 2, was determined to be S/Q G K Q Y S EX G/S K, denoted SEQ ID NO:27. This amino acid sequence was essentiallythe same, or at least a subset of, the N-terminal amino acid sequenceobtained from flea saliva protein fspA.

Sequence analysis of Peak G proteins indicated the presence of threeproteins in that peak, referred to herein as fspG1, fspG2 and fspG3.Flea saliva protein fspG1, having a molecular weight of about 9 kD, hadan N-terminal partial amino acid sequence of D R R V S K, denoted SEQ IDNO:28. This N-terminal amino acid sequence is the same as that for fspH,as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:14. Flea saliva protein fspG2, having a molecular weight of about7 kD, had an N-terminal partial amino acid sequence of S K M V T E K X KS G G N N P S T K E V S I P, denoted SEQ ID NO:29. Flea saliva proteinfspG3, having a molecular weight of about 6 kD, had an N-terminalpartial amino acid sequence of E V S I P S G K L T I E D F X I G N H Q,denoted SEQ ID NO:30. A comparison of SEQ ID NO:30 with SEQ ID NO:29indicates that fspG3 may be a proteolytic degradation product of fspG2,as the last five amino acids of fspG2 are identical with those at theN-terminus of fspG3.

The N-terminal partial amino acid sequence of flea saliva protein fspH,which migrated as Peak H in FIG. 2, was determined to be D R R V S K T XQ S G G K I Q S E X Q V V I K S G Q H/Y I L E N Y X S D G R, denotedherein as SEQ ID NO:14. Histidine and tyrosine were equally likely atamino acid position 27.

Flea saliva protein fspH was also subjected to proteolytic cleavage inorder to obtain internal amino acid sequence data. Specifically, fspHwas cleaved with Endoproteinase Asp-N (available from BoehringerMannheim Biochemica, Indianapolis, Ind.) using methods standard in theart. The digested protein was then resolved by HPLC using the methoddescribed by Stone et al. (ibid.). The resultant HPLC profile is shownin FIG. 3. Three proteolytic fragments were isolated, that are referredto herein as fspHe, fspHh and fspHj.

The N-terminal partial amino acid sequence of fspHe was determined to beD S K H C Y C E A P Y S, also denoted SEQ ID NO:3. The N-terminalpartial amino acid sequence of fspHh was determined to be D G R N N N NP C H L F C M R E C R S G N G G C G N G G R T R P D S K H C, alsodenoted SEQ ID NO:4. The N-terminal partial amino acid sequence of fspHjwas determined to be D R R V S K T C Q S G, also denoted SEQ ID NO:5.Comparison of SEQ ID NO:5 to SEQ ID NO:14 indicated that fspHj was theN-terminal fragment of fspH.

By aligning SEQ ID NO:14, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, thefollowing amino acid sequence was deduced, starting at the N-terminus offspH: D R R V S K T C Q S G G K I Q S E X Q V V I K S G Q H/Y I L E N YX S D G R N N N N P C H L F C M R E C R S G N G G C G N G G R T R P D SK H C Y C E A P Y S. This amino acid sequence is denoted SEQ ID NO:2 andis believed to represent most of fspH since the molecular weight of aprotein having this sequence is about 8600 kD.

The N-terminal partial amino acid sequence of flea saliva protein fspI,which migrated as Peak I in FIG. 2, was determined to be E D I W K V N KK X T S G G K N Q D R K L D Q I I Q K G Q Q V X X Q N X X K, denotedherein as SEQ ID NO:6.

Sequence analysis of Peak J proteins indicated the presence of twoproteins in that peak, referred to herein as fspJ1 and fspJ2. TheN-terminal partial amino acid sequence of flea saliva protein fspJ1 wasdetermined to be N S H E P G N T R K I R E V M D K L R K Q H P, denotedherein as SEQ ID NO:7. The N-terminal partial amino acid sequence offlea saliva protein fspJ2 was determined to be E I K R N S H E P G N T RK I R E V M D K L R K Q H P, denoted herein as SEQ ID NO:8. The proteinsrepresented by SEQ ID NO:7 and SEQ ID NO:8 were not separately resolvedby SDS-PAGE as described in Example 1. Comparison of SEQ ID NO:7 and SEQID NO:8 suggest that fspJ1 may be a truncated version of fspJ2, in thatthe N-terminal partial amino acid sequence of fspJ1 appears to be verysimilar to that of fspJ2 except that fspJ1 lacks the first 4 amino acidsfound at the N-terminus of fspJ2.

Sequence analysis of Peak L proteins indicated the presence of twoproteins in that peak, referred to herein as fspL1 and fspL2. That therewere two proteins, namely fspL1 and fspL2, was shown by subjecting peakL to C4 reverse phase chromatography using 0.13% heptafluorobutyric acid(Solvent A) and 0.1% heptafluorobutyric acid in 90% acetonitrile(Solvent B) in the following gradient format: an 80 minute gradient from30% Solvent B to 70% Solvent B. The N-terminal partial amino acidsequence of the HPLC-separated fspL1 was determined to be N D K E P G NT R K I R E V M D K L R K Q A Q P R T D G Q R P K T X I M, also denotedSEQ ID NO:9. The N-terminal partial amino acid sequence for fspL2 wasdetermined to be X L X R N D K E P G N T R K I R E V M D K, also denotedSEQ ID NO:10. A comparison of SEQ ID NO:9 and SEQ ID NO:10 indicatesthat fspL1 and fspL2 are similar proteins, except that fspL1 is 4 aminoacids shorter than fspL2 at the N-terminus.

Resolution of proteins contained in Peak N by SDS-PAGE as described inExample 3 revealed 3 distinct bands. The bands were denoted flea salivaproteins fspN1, fspN2 and fspN3. The N-terminal partial amino acidsequence of fspN1 was determined to be N D E L K F V F V M A K, alsodenoted SEQ ID NO:11. The N-terminal partial amino acid sequence offspN2 was determined to be X D E L K F V F V M A K G P S X Q A X D Y PC, also denoted SEQ ID NO:12. The N-terminal partial amino acid sequenceof fspN3 was determined to be E L K F V F A T A R G M S H T P C D Y P,also denoted SEQ ID NO:13. Comparison of SEQ ID NO:11 and SEQ ID NO:12suggests that fspN1 and fspN2 share the same N-terminal sequence. SincefspN1 and fspN2 migrate differently when subjected to SDS-PAGE, however,the two proteins are likely to be different homologues, possibly due toone protein having a longer C-terminal domain and/or due topost-translational modification(s). Comparison of SEQ ID NO:13 to SEQ IDNO:11 and SEQ ID NO:12 suggests that fspN3 may be a homologue of fspN1and fspN2 with internal sequence variations.

Flea saliva proteins in Peak N were also subjected to proteolyticcleavage in order to obtain internal amino acid sequence data.Specifically, the proteins in Peak N were cleaved with EndoproteinaseAsp-N (available from Boehringer Mannheim Biochemica, Indianapolis,Ind.) using methods standard in the art. The digested protein was thenresolved by HPLC using the method described by Stone et al. (ibid.) andsequenced as previously described. A partial amino acid sequence of fleasaliva proteins in Peak N, named fragment pfspN(100-101), was determinedto be D I E N I K K G E G Q P G A P G G K E N N L S/L V L, denotedherein as SEQ ID NO:31.

Example 5

This example describes the further characterization of proteinscontained in Peak H.

To determine the isoelectric pH of the proteins contained in Peak H,proteins present in that peak were resolved using standard isoelectricfocusing techniques known to those of skill in the art; see, forexample, O'Farrell, 1975, J. Biol. Chem., Vol. 250, pp. 4007-4021. ThepI value for proteins contained in Peak H is about pI 9, ranging fromabout pI 8.5 to about pI 9.6.

The molecular weight of proteins contained in Peak H was determined byESMS. The ESMS procedure was performed on a Fisons VG quattro-SQ massspectrometer. The mass range was calibrated for 100-2000 m/z. Theinjection rate was performed at 4 μl per minute. The cone voltage wasset at 45 volts. The injection sample contained 0.1% formic acid in 50%acetonitrile at a protein concentration of about 100 pmole per μl. Theresults indicate that Peak H apparently contains a population ofproteins all having a molecular weight of 8613±6 daltons.

Example 6

This example describes the isolation of nucleic acid sequences encodingat least portions of flea saliva proteins fspH and fspI.

A. Description of Flea Libraries

Fed flea and unfed flea cDNA libraries were prepared using standardprocedures. Briefly, about 3000 to 4000 fed fleas and about the samenumber of unfed fleas were collected separately, placed into a dry-icecooled mortar/pestle and ground to a fine powder. RNA from the ground-upfleas was prepared by direct extraction using the guanidiniumthiocyanate procedure followed by centrifugation in cesium chloridegradients (see, for example, Sambrook et al., ibid.). Cesiumchloride-purified gelatinous RNA pellets were collected and dissolved insterile TE buffer (10 mM Tris-HCl, pH 7.6 and 1 mM EDTA) containing 0.1%sodium dodecyl sulfate. The dissolved pellet was precipitated withaddition of 3 M sodium acetate, pH 5.2 to a final concentration of 0.2mM and two volumes of absolute ethanol to remove residual CsCl. TotalRNA was fractionated for enrichment of the mRNA fraction using aFastTrack™ kit (available, with procedures, from InVitrogen Corp., SanDiego, Calif.).

Isolated whole flea mRNA was used directly for cDNA synthesis andmolecular cloning. The methods of cDNA synthesis and molecular cloningwere those provided with the Lambda Zap-cDNA synthesis Kit™ (availablefrom Stratagene, Inc., La Jolla, Calif.). Following is a list of fleacDNA libraries prepared having the indicated number of total plaqueforming units (PFU) packaged: (a) two whole fed flea cDNA expressionlibraries referred to as Library C (about 2.5×10⁶ PFU) and Library H(about 1.3×10⁶ PFU); (b) a whole unfed flea cDNA expression library(about 1.3×10⁶ PFU); (c) a flea salivary gland cDNA expression libraryprepared from approximately 6000 salivary glands collected from fed andunfed fleas (about 1.5×10⁶ PFU); and (d) a flea fed midgut cDNAexpression library prepared from approximately 5000 isolated midguts(about 2.3×10⁶ PFU).

B. Isolation of a Nucleic Acid Molecule Encoding fspH

A nucleic acid molecule encoding a portion of flea saliva protein fspHwas identified using the flea salivary gland cDNA expression librarydescribed in Example 6A.

Degenerate synthetic oligonucleotide primers were designed from theamino acid sequence deduced for fspH (see Example 4). Three syntheticoligonucleotides were synthesized that corresponded to the region offspH spanning from about residues 38 through 51 of SEQ ID NO:2: Primer1, a “sense” primer corresponding to amino acid residues from about 38through about 44 of SEQ ID NO:2, has the nucleic acid sequence 5′ AAT(C)AAT(C) AAT(C) AAT(C) CCT(GAC) TGT(C) CA 3′, and is denoted SEQ ID NO:15.Primer 2, an “antisense” primer corresponding to amino acid residuesfrom about 46 through about 51 of SEQ ID NO:2, has the nucleic acidsequence 5° CA C(T)TC C(TAG)CT(G) CAT G(A)CA G(A)AA 3′ and is denotedSEQ ID NO:16. Primer 3, a sense primer corresponding to amino acidresidues from about 43 through about 48 of SEQ ID NO:2, has the nucleicacid sequence 5′ TGT(C) CAT(C) T(C)TG(ATC) TTT(C) TGC(T) ATG-3′ and isdenoted SEQ ID NO:17. A fourth primer, Primer 4, was synthesized thatcorresponded to the carboxyl region of fspH, namely spanning from aboutamino residue 69 through 76 of SEQ ID NO:2. Primer 4, an antisenseprimer, has the nucleic acid sequence 5′ GGA(CGA) GCT(C) TCA(G) CAA(G)TAA(G) CAA(G) TGT(C) TT′ 3′, denoted SEQ ID NO:18.

PCR amplification of fragments from the flea salivary gland library wasconducted using standard techniques. PCR amplification products weregenerated using the combination of Primer 1 and the M13 forwarduniversal standard primer 5′ GTAAAACGACGGCCAGT 3′, denoted SEQ ID NO:19.The resultant PCR amplification products were used for a nested PCRamplification using Primer 3 and Primer 4. The resultant PCR product, afragment of 101 nucleotides denoted nfspH₁₀₁, was cloned into theInVitrogen, Corp., TA™ cloning vector (procedures provided byInVitrogen, Corp.) and subjected to DNA sequence analysis using standardtechniques. The resulting nucleic acid sequence is presented as SEQ IDNO:20:

T TGT CAC TTT TTT TGT ATG AGA GAA TGC AGG TCA GGA AAC GGC GGT TGC GGAAAC GGA GGA AGG ACA AGA CCT GAT TCG AAG CAC TGC TAT GC(primer-derived sequences are in bold). The 60 nucleotides of internalnon-primer-derived sequence codes for 20 amino acids of fspH, spanningfrom residue about 48 through about 68, as numbered in SEQ ID NO:2.

Using standard techniques, nucleic acid molecule nfspH₁₀₁ can be used asa probe to isolate a nucleic acid molecule that encodes a proteincorresponding to a full-length, or larger partial, fspH protein.

C. Isolation of a Nucleic Acid Molecule Encoding fspI

The amino acid sequence for fspI (SEQ ID NO:6) disclosed in Example 4was used to design a set of synthetic degenerate oligonucleotide PCRamplification primers. Degenerate Primer 5, a sense primer correspondingto residues from about 1 through about 8 of SEQ ID NO:6, has the nucleicacid sequence 5′ GAA(G) GAT(C) ATT(CA) TGG AAA(G) GTT(CAG) AAT(C) AA 3′,denoted SEQ ID NO:21. Degenerate Primer 6, a sense primer correspondingto residues from about 11 through about 18 of SEQ ID NO:6, has thenucleic acid sequence 5′ ACT(CGA) TCT(CGA) GGT(CGA) GGT(CGA) AAA(G)AAT(C) CAA(G) GA 3′, denoted SEQ ID NO:22.

Primers 5 and 6 were used in combination with the vector primers BSKX(5′ TTGGGTACCGGGCCCCCCCT 3′, SEQ ID NO:23) and the M13 primer denoted bySEQ ID NO:19 in order to generate PCR amplification products. The PCRproducts were cloned into the InVitrogen TA™ vector and subjected to DNAsequence analysis. One cloned product analyzed, called nfspI₅₇₃contained a 573-nucleotide product that corresponded, at least in part,to the partial amino acid sequence determined for fspI. The nucleotidesequence of nfspI₅₇₃ is presented as SEQ ID NO:24. Translation of SEQ IDNO:24 yields the following longest open reading frame, denoted as SEQ IDNO:25.

By combining the partial N-terminal sequence of fspI (SEQ ID NO:6) withthe protein sequence SEQ ID NO:25 deduced from the nucleic acid sequenceSEQ ID NO:24, it is possible to obtain an apparent full-length aminoacid sequence for fspI, denoted SEQ ID NO:26.

Example 7

This example further describes the isolation of nucleic acid sequencesencoding at least portions of flea saliva proteins fspH and fspI.

A. Amplification of a Nucleic Acid Molecule Encoding fspH

The DNA sequence determined from the carboxyl-terminal PCR product (SEQID NO:20) in Example 6B was used to synthesize two non-degeneratesynthetic homologous primers: Primer 7, 5′ CCT GAC CTG CAT TCT CTC ATA C3′, denoted SEQ ID NO:38, and Primer 8, 5′ AGG TCT TGT CCT TCC TCC GTTTCC GCA 3′, denoted SEQ ID NO:39. Primer 8 was used in combination withthe M13 reverse primer 5′ GGAAACAGCTATGACCATG 3′, denoted SEQ ID NO:40,to amplify the 5′-terminal portion of the fspH gene from a fraction ofthe salivary gland cDNA expression library described above in Example 6Ausing standard techniques. The resultant PCR product, although notclearly visible on a gel, was identified as a single product by Southernhybridization using Primer 7 as a [³²P]-radiolabeled probe. A clearlyvisible ethidium bromide stained PCR product was obtained by performinga nested PCR reaction utilizing Primer 7 and the vector T3 primer, 5′ATTAACCCTCACTAAAG 3′, denoted SEQ ID NO:41. The approximately 400-bpproduct was clearly visible on a 1% agarose gel and washybridization-positive with [³²P]-labeled degenerate Primer 1 (SEQ IDNO:15).

A partial, 242-bp, nucleotide sequence of the 400-bp product of fspH,named nfspH₂₄₂ is presented as SEQ ID NO:32. Translation of SEQ ID NO:32yields the amino acid sequence, named PfspH₈₀ denoted as SEQ ID NO:33.

B. Amplification of a Nucleic Acid Molecule Encoding fspI

Two additional primers were made for isolating the fspI protein cDNAsequence from the flea salivary gland library prepared as described inExample 6A. The isolation procedure used PCR reiteration of the fleasalivary gland library and probe hybridization to the PCR generatedproducts. Repeating the PCR on fractions of the flea salivary glandlibrary (mini-libraries) narrowed the occurrence frequency of the clonedfspI protein cDNA to approximately 1 in 200 plaque forming units (PFU)before a final plaque lift and identification by hybridization with a[³²P]-labeled probe.

Two primers based on SEQ ID NO:24 were used: Primer 9, 5′ GCA AAG GTTATA GAG GAG CTT G 3′, denoted as SEQ ID NO:42, and Primer 10, 5′ AGC TTTCCA TCA CAT CCA GC 3′, denoted as SEQ ID NO:43. The primers generated aninternal PCR DNA sequence of 268 bp (including primers) which was usedas a marker sequence for screening the salivary gland mini-libraries.The final screening of the salivary gland mini-libraries was done with apool of the four [³²P]-labeled primers; Primer 5, SEQ ID NO:21, Primer6, SEQ ID NO:22, Primer 8, SEQ ID NO:42 and Primer 10, SEQ ID NO:43,using standard techniques. A nucleic acid molecule, named nfspI₅₉₁,identified by this technique was sequenced using standard techniques togive SEQ ID NO:34. The translation of SEQ ID NO:34 yielded yields theamino acid sequence, denoted as SEQ ID NO:35, for a protein namedPfspI₁₅₅. This amino acid sequence is similar to SEQ ID NO:26, exceptthat SEQ ID NO:35 does not contain the amino acid sequence E D I at theamino terminus, and SEQ ID NO:35 contains a “C” at position 7, whereasSEQ ID NO:26 has an “L” at the corresponding position.

Example 8

This example demonstrates the ability of a formulation of the presentinvention to induce flea allergy dermatitis in an animal susceptible toflea allergy dermatitis.

To determine whether the isolated flea saliva proteins described inExamples 2 and 3 were capable of inducing an allergic response inanimals susceptible to flea allergy dermatitis, skin tests wereperformed on sensitized dogs. Six dogs were sensitized to fleas usingthe method of Gross, et al., 1985, Veterinary Pathology, Vol. 22, pp.78-71. Briefly, each dog was exposed to about 25 C. felis fleascontained in chambers by allowing the contained fleas to feed on theexperimental dogs for about 15-minute periods at weekly intervals. Thesix dogs were sensitized over the following periods: Dog 2080109 wasexposed to fleas 38 times over a period spanning Aug. 31, 1993 throughJun. 7, 1994. Dog 2082101 was exposed to fleas 22 times over a periodspanning Dec. 14, 1993 through Jun. 7, 1994. Dog 2082128 was exposed tofleas 20 times over a period spanning Aug. 31, 1993 through May 24,1994. Dog BFQ2 was exposed to fleas 17 times over a period spanning Mar.15, 1994 through Jul. 12, 1994. Dog CPO2 was exposed to fleas 12 timesover a period spanning Mar. 15, 1994 through Jun. 7, 1994. Dog CQQ2 wasexposed to fleas 1 time on Mar. 15, 1994.

Skin testing was performed the morning of Jul. 21, 1994. The dogs wereshaved in the lateral thorax/abdominal area and intradermally injectedin that area with a variety of formulations of the present invention aswell as with control solutions. The total volume per injection was 50microliters (μl), with the formulations and controls being diluted inphenolated saline. Each dog received the injections listed in Table 1.

TABLE 1 Samples administered to dogs. SAMPLE REPLICATES μg/inj FLEA-HOURDILUENT 2 N/A* N/A HISTAMINE 2 1.38 N/A GREER 3 50 (w/v) N/A FS-1 3 1.884,660 A 3 0.23 23,000 B 3 0.32 23,000 C1 3 1.10** 23,000 C2 3 0.4223,000 D1 3 0.24 23,000 D2 3 0.29 23,000 E 3 0.16 23,000 F 3 0.10 23,000G 3 0.21 23,000 H 3 0.20 23,000 I 3 0.12 23,000 J 3 0.08 23,000 K 3 0.1223,000 L 3 0.08 23,000 M1 3 0.16 23,000 M2 3 0.27 23,000 N 3 0.20 23,000FS-2 3 0.71 4,660 *N/A is not applicable **Apparent amount, probablyartificially high due to assay interferenceNote that in these studies, fspJ1 and fspJ2 were administered togetheras fspJ; fspL1 and fspL2 were administered together as fspL; fspN1,fspN2 and fspN3 were administered together as fspN. It is also to benoted that A, B, C1, C2, D1, D2, E, F, G, H, I, J, K, L, M1, M2 and Nrefer, respectively to flea saliva proteins fspA, fspB, fspC1, fspC2,fspD1, fspD2, fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1,fspM2 and fspN. The negative control comprised diluent (NC) and thepositive controls comprised Greer antigen (GR) and histamine (HIS). Theamount of Greer antigen used was determined by weight per volume (w/v)according to the information provided by the manufacturers (GreerLaboratories, Inc., Lenoir, N.C.). The amount of histamine used wasdetermined by information provided on the supplier's label (availablefrom Greer Laboratories, Inc., Lenoir, N.C.).A. Comparison of Wheal Sizes at Sites of Injection

All injection sites were objectively (Obj) measured in millimeters (mm)at 15 min and subjectively (Sub) scored on a scale of 0 to 4. Thesubjective scoring was performed by Kenneth W. Kwochka, D.V.M., DiplomatACVD, (American College of Veterinary Dermatologists) at Ohio StateUniversity, Columbus, Ohio. Tables 2 through 7 indicate the resultsobtained for each dog. # refers to the number designation given to eachsample; antigen refers to the sample. Inj 1, Inj 2 and Inj 3 refer totriplicate injections and NA refers to “not applicable.”

TABLE 2 DOG ID: 2082101 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # AntigenSub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 4 12 NANA NA NA 3 Greer 3 10 3 10 3 10 4 FS-1 3 10 4 12 4 12 5 A 1 8 0 8 0 8 6B 0 6 0 6 0 6 7 C1 0 6 0 6 0 6 8 C2 0 6 0 6 0 6 9 D1 0 8 0 8 0 6 10 D2 06 0 6 0 8 11 E 3 12 3 12 3 12 12 F 3 14 3 12 3 12 13 G 3 12 3 12 3 12 14H 3 11 2 12 3 12 15 I 3 12 2 12 3 11 16 J 2 10 2 11 2 10 17 K 2 11 2 102 9 18 L 2 9 1 10 1 10 19 M1 2 12 2 11 2 11 20 M2 3 12 3 11 3 12 21 N 311 3 10 2 11 22 FS-2 2 11 3 12 2 10 23 Neg Cntl 0 8 NA NA NA NA 24Histamine 4 14 NA NA NA NA

TABLE 3 DOG ID: 2080109 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # AntigenSub Obj Sub Obj Sub Obj 1 Neg Cntl 0 7 NA NA NA NA 2 Histamine 4 14 NANA NA NA 3 Greer 0 8 0 8 0 8 4 FS-1 4 13 4 13 4 13 5 A 0 9 0 8 0 8 6 B 07 0 7 0 7 7 C1 0 8 0 7 0 7 8 C2 0 8 0 7 0 8 9 D1 1 9 1 9 1 9 10 D2 1 9 18 1 8 11 E 3 11 3 11 2 10 12 F 3 11 3 13 4 13 13 G 3 14 3 13 3 13 14 H 212 2 11 2 10 15 I 2 10 3 10 3 10 16 J 2 10 3 10 3 10 17 K 2 9 2 9 2 9 18L 1 9 1 6 1 7 19 M1 3 11 3 13 3 13 20 M2 3 14 3 13 3 14 21 N 3 13 3 14 210 22 FS-2 2 9 1 7 1 8 23 Neg Cntl 0 6 NA NA NA NA 24 Histamine 4 16 NANA NA NA

TABLE 4 DOG ID: 2082128 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # AntigenSub Obj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 4 12 NANA NA NA 3 Greer 0 6 0 6 0 6 4 FS-1 3 12 3 12 3 12 5 A 0 7 0 7 0 6 6 B 07 0 7 0 6 7 C1 0 7 0 6 0 7 8 C2 0 6 0 7 0 7 9 D1 0 7 0 7 0 7 10 D2 0 7 07 0 7 11 E 0 7 0 6 0 7 12 F 0 6 0 6 0 6 13 G 1 10 1 9 1 9 14 H 2 10 2 102 11 15 I 3 12 3 12 3 11 16 J 3 12 3 11 3 11 17 K 3 11 3 12 3 12 18 L 311 3 10 3 11 19 M1 3 11 3 11 3 12 20 M2 3 12 3 12 3 12 21 N 3 12 3 12 312 22 FS-2 3 12 3 11 3 12 23 Neg Cntl 0 6 NA NA NA NA 24 Histamine 4 14NA NA NA NA

TABLE 5 DOG ID: BFQ2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # Antigen SubObj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 4 12 NA NA NANA 3 Greer 0 6 0 6 0 6 4 FS-1 1 9 1 9 1 9 5 A 0 7 0 7 0 7 6 B 0 7 0 7 07 7 C1 0 7 1 7 1 7 8 C2 0 7 0 7 0 6 9 D1 0 8 1 7 1 8 10 D2 0 7 0 6 1 711 E 1 7 0 6 0 6 12 F 1 6 1 7 0 7 13 G 0 8 1 8 1 8 14 H 0 8 0 7 0 7 15 I1 7 0 7 0 8 16 J 0 7 0 7 0 7 17 K 0 7 0 7 0 6 18 L 0 8 0 7 0 7 19 M1 0 70 7 0 7 20 M2 0 7 0 7 1 8 21 N 3 12 3 11 3 11 22 FS-2 3 11 3 11 3 11 23Neg Cntl 0 7 NA NA NA NA 24 Histamine 4 15 NA NA NA NA

TABLE 6 DOG ID: CPO2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # Antigen SubObj Sub Obj Sub Obj 1 Neg Cntl 0 3 NA NA NA NA 2 Histamine 4 13 NA NA NANA 3 Greer 0 7 0 7 0 6 4 FS-1 4 12 4 12 4 12 5 A 0 7 0 6 0 6 6 B 0 6 0 70 7 7 C1 0 7 0 6 0 7 8 C2 0 6 0 6 0 6 9 D1 0 7 1 7 0 7 10 D2 1 6 0 6 0 511 E 0 6 0 6 0 6 12 F 0 6 0 6 2 7 13 G 2 9 2 8 2 8 14 H 4 11 4 12 4 1115 I 3 12 3 11 3 10 16 J 3 10 3 11 3 10 17 K 2 8 2 8 2 8 18 L 1 8 1 7 17 19 M1 3 11 3 11 3 11 20 M2 3 11 4 12 4 12 21 N 4 12 3 10 3 11 22 FS-23 11 3 12 3 12 23 Neg Cntl 0 6 NA NA NA NA 24 Histamine 4 13 NA NA NA NA

TABLE 7 DOG ID: CQQ2 Inj 1 Inj 1 Inj 2 Inj 2 Inj 3 Inj 3 # Antigen SubObj Sub Obj Sub Obj 1 Neg Cntl 0 6 NA NA NA NA 2 Histamine 4 13 NA NA NANA 3 Greer 0 7 0 7 0 7 4 FS-1 2 8 2 8 2 8 5 A 0 6 0 6 0 7 6 B 0 7 0 7 06 7 C1 0 7 0 6 0 6 8 C2 0 7 0 7 0 6 9 D1 0 6 0 6 0 6 10 D2 0 6 0 6 0 711 E 0 6 0 6 0 6 12 F 0 6 0 7 0 7 13 G 0 7 0 7 0 6 14 H 1 7 1 7 1 7 15 I2 8 2 9 2 8 16 J 2 8 2 8 2 8 17 K 1 7 1 7 1 7 18 L 1 6 0 6 0 6 19 M1 2 72 8 2 8 20 M2 2 8 2 8 2 9 21 N 3 11 3 12 3 11 22 FS-2 3 11 3 11 3 10 23Neg Cntl 0 7 NA NA NA NA 24 Histamine 4 14 NA NA NA NA

As a control, 2 flea naive dogs (i.e., dogs that had never been exposedto fleas) were also tested with single replicates of the same samplesthat were injected into the sensitized dogs. These dogs are referred toas WANU and WBCE. Objective and subjective wheal size measurements 15minutes after injection of the samples are shown in Tables 8 and 9.

TABLE 8 DOG ID: WANU Inj 1 Inj 1 # Antigen Sub Obj 1 Neg Cntl 0 7 2Histamine 4 10 3 Greer 0 6 4 FS-1 0 6 5 A 0 7 6 B 0 6 7 C1 0 6 8 C2 0 69 D1 0 7 10 D2 0 6 11 E 0 6 12 F 0 6 13 G 0 7 14 H 0 7 15 I 0 7 16 J 0 717 K 0 6 18 L 0 7 19 M1 0 6 20 M2 0 6 21 N 1 8 22 FS-2 1 8 23 Neg CntlNA NA 24 Histamine NA NA

TABLE 9 DOG ID: WBCE Inj 1 Inj 1 # Antigen Sub Obj 1 Neg Cntl 0 6 2Histamine 4 12 3 Greer 0 7 4 FS-1 0 7 5 A 0 7 6 B 0 7 7 C1 0 7 8 C2 0 79 D1 0 7 10 D2 0 6 11 E 0 7 12 F 0 7 13 G 0 8 14 H 0 7 15 I 0 7 16 J 0 717 K 0 7 18 L 0 6 19 M1 0 7 20 M2 0 7 21 N 0 7 22 FS-2 0 7 23 Neg CntlNA NA 24 Histamine NA NA

The average subjective score obtained for each flea saliva antigen fromthe 6 sensitized dogs tested was calculated and is summarized in FIG. 5.The results indicate that the flea saliva products produced as describedin Examples 2 and 3 include at least one allergenic protein capable ofinducing an immediate hypersensitive response in a sensitized dog. Inparticular, injection of the mixtures of flea saliva antigens referredto as FS-1 and FS-2 resulted in substantial wheal formation. Flea salivaproteins fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1, fspM2and fspN also resulted in substantial wheal formation. Flea salivaproteins fspA, fspB, fspC1, fspC2, fspD1 and fspD2 produced minimal, ifany, allergic response, depending on the dog being tested. The samplecontaining fspH produced the largest wheal formation when compared withthe other flea saliva proteins.

*B. Comparison of Levels of Induration and Erythema at the InjectionSites

In addition to wheal size, the amount of induration and erythema werealso measured at each site of injection. Induration produced by theinjection of the flea saliva antigens was measured at 6 hours and 24hours by subjective scoring. Such subjective induration measurementswere performed by Kenneth W. Kwochka, D.V.M. In addition, the amount oferythema at each site of injection were subjectively scored by KennethW. Kwochka, D.V.M.

The amounts of induration and erythema measured by subjective scoring at6 hours were negative for each of the sensitized and control dogs exceptfor the following formulations in the following sensitized dogs.Administration of FS-1 to Dog 2082101 produced an average indurationscore of 1 at 2 sites of injection but no erythema score. Administrationof fspL to Dog 2082101 produced no induration but an erythema score of 1at 1 site of injection. Administration of fspM1 to Dog 2082101 producedno induration but an erythema score of 3 at 1 site of injection.Administration of FS-2 to Dog 2082101 produced no induration but anaverage erythema score of 1.33 at 3 sites of injection. Administrationof fspH to Dog 2082128 produced no induration but an average erythemascore of 2 at 3 sites of injection. Administration of fspI to Dog2082128 produced an average induration score of 1 and an averageerythema score of 1 at 2 sites of injection. Administration of fspJ toDog 2082128 produced an average induration score of 1 and an averageerythema score of 1 at 3 sites of injection. Administration of FS-2 toDog 2082128 produced no induration but an average erythema score of 2 at3 sites of injection.

Administration of FS-1 to Dog BFQ2 produced an average induration scoreof 2 and an average erythema score of 2 at 3 sites of injection.Administration of fspN to Dog BFQ2 produced an average induration scoreof 1 and an average erythema score of 2 at 2 sites of injection.Administration of FS-2 to Dog BFQ2 produced an average induration scoreof 1 and an average erythema score of 2 at 2 sites of injection.

Administration of FS-1 to Dog CPO2 produced an average induration scoreof 2.5 but no erythema at 2 sites of injection. Administration of fspGto Dog CPO2 produced no induration but an average erythema score of 2 at3 sites of injection. Administration of fspH to Dog CPO2 produced noinduration but an average erythema score of 1 at 2 sites of injection.Administration of FS-2 to Dog CPO2 produced no induration but an averageerythema score of 2 at 3 sites of injection.

The average subjective score for induration obtained for each fleasaliva antigen from the 6 sensitized dogs tested was calculated and issummarized in FIG. 6. The average subjective score for erythema obtainedfor each flea saliva antigen from the 6 sensitized dogs tested wascalculated and is summarized in FIG. 7.

The amounts of induration and erythema measured by subjective scoring at24 hours results for five of the flea-sensitized dogs and the twocontrol dogs were negative except for the following formulations in thefollowing sensitized dogs.

Administration of fspI to Dog 2082101 produced an average indurationscore of 1 and an average erythema score of 1 at 3 sites of injection.Administration of fspJ to Dog 2082101 produced an average indurationscore of 1 and an average erythema score of 1 at 3 sites of injection.Administration of fspM1 to Dog 2082101 produced an average indurationscore of 1 and an average erythema score of 3 at 3 sites of injection.Administration of fspN to Dog 2082101 produced an average indurationscore of 1 and an average erythema score of 2 at 3 sites of injection.Administration of FS-2 to Dog 2082101 produced an average indurationscore of 3 and an average erythema score of 4 at 3 sites of injection.

Administration of FS-1 to Dog BFQ2 produced an average induration scoreof 1 and an average erythema score of 1 at 3 sites of injection.Administration of FS-2 to Dog BFQ2 produced an induration score of 1 andan erythema score of 1 at 1 site of injection.

Administration of FS-1 to Dog CPO2 produced an induration score of 2 andan erythema score of 1 at 1 site of injection. Administration of fspI toDog CPO2 produced an average induration score of 1 and an averageerythema score of 1 at 3 sites of injection. Administration of FS-2 toDog CPO2 produced an average induration score of 1 and an averageerythema score of 2 at 3 sites of injection.

Administration of Greer antigen to Dog CQQ2 produced no induration butan average erythema score of 1 at 3 sites of injection. Administrationof FS-1 to Dog CQQ2 produced an induration score of 1 and an erythemascore of 1 at 1 site of injection. Administration of fspI, fspJ, fspM1or fspM2 to Dog CQQ2 produced no induration but an average erythemascore of 1 at 3 sites of injection. Administration of fspN to Dog CQQ2produced an induration score of 1 and an erythema score of 1 at 1 siteof injection. Administration of FS-2 to Dog CQQ2 produced an averageinduration score of 1 and an average erythema score of 2 at 3 sites ofinjection.

The average subjective score for induration obtained for each fleasaliva antigen from the 6 sensitized dogs tested was calculated and issummarized in FIG. 8. The average subjective score for erythema obtainedfor each flea saliva antigen from the 6 sensitized dogs tested wascalculated and is summarized in FIG. 9.

The results indicate that at least some of the flea saliva proteinformulations produced as described in Examples 2 and 3 include at leastone allergenic protein capable of inducing a delayed hypersensitiveresponse in a sensitized dog. Injection of the mixtures of flea salivaproteins referred to as FS-1 and FS-2 induced substantial induration anderythema for at least 24 hours. In addition, the flea saliva proteinsamples fspI, fspJ, M1 and fspN were sufficiently allergenic to induceinduration and erythema for at least 24 hours. The flea saliva proteinsample fspL and fspM2 induced substantial levels of induration but notsubstantial levels of erythema at 24 hours.

Taken together, the results shown indicated above and shown in FIG. 5through 9, indicate that saliva protein formulations of the presentinvention are sufficiently allergenic to induce a hypersensitiveresponse in a sensitized dog. Numerous samples induced both an immediatehypersensitive response and a delayed hypersensitive response.

Example 9

This example demonstrates the ability of numerous flea saliva proteinsamples isolated in Examples 2 and 3 to induce a hypersensitive responseby histopathology of tissue removed from selected lesions on the dogsdescribed in Example 8.

Two tissue samples per dog were removed from each sensitized dogdescribed in Example 8. No biopsies were taken from the two naive dogs.The selected sites from which the tissue samples were removed areindicated in Table 10 below. Biopsies were taken with a 4 mm biopsypunch after subcutaneous injections of Lidocaine. Biopsies wereprocessed and read by Dr. David M. Getzy, DVM, Diplomat ACVP (AmericanCollege of Veterinary Pathologists) at the Colorado VeterinaryDiagnostic Laboratory (College of Veterinary Medicine and BiomedicalSciences, Colorado State University, Fort Collins, Colo.).

TABLE 10 Histopathology Dog Antigen Time No. Slide Lesion Type Grade 101FS-1 15 min. 1 A A 1 6 hr. 2 B A 2.5 24 hr. 3 C A 3 109 FS-1 15 min. 4 DA 1 6 hr. 5 E C 2 24 hr. 6 F C 3 128 FS-1 15 min. 7 G A 1.5 6 hr. 8 H C1.5 24 hr. 9 I C 3 CPO2 FS-1 15 min. 10 J A 1.5 6 hr. 11 K C 3 24 hr. 12L C 4 CQQ2 FS-1 15 min. 13 M A 1.5 6 hr. 14 N C 2.5 24 hr. 15 O C 2.5101 fspE 15 min. 16 P A 1 6 hr. 17 Q C 1.5 24 hr. 18 R A 1.5 109 fspF 15min. 19 S A 1 6 hr. 20 T A 1.5 24 hr. 21 U A 1.5 128 fspI 15 min. 22 V A1 6 hr. 23 W C 2.5 24 hr. 24 X C 2.5 BFQ2 fspN 15 min. 25 Y A 1.5 6 hr.26 Z C 2 24 hr. 27 AA C 3.5 BFQ2 fspO 15 min. 28 BB A 1 6 hr. 29 CC C 324 hr. 30 DD C 2.5 CPO2 fspH 15 min. 31 EE A 1.5 6 hr. 32 FF C 1.5 24hr. 33 GG A 1.5 CQQ2 fspN 15 min. 34 HH A 1 6 hr. 35 II C 2.5 24 hr. 36JJ C 2.5

Two types of lesions were found in the tissue samples tested. LesionType A refers to a moderate superficial dermal edema having mild numbersof mast cells in a perivascular orientation within the superficialdermis. Vascular endothelium exhibited mild reactive hypertrophy.Minimal numbers of neutrophils were noted in this region as well. LesionType C refers to lesions that were similar to those described in LesionType A except that the eosinophils were mild to moderate in severity,while neutrophils and mast cells were mild in severity.

On a scale of 0 to 5, lesions ranged from grade 1 to grade 4 inseverity. Some of the specimens had predominantly mastocyticinflammatory perivascular infiltrates, edema, and minimal numbers ofother cellular components. Other sections showed a predominance ofeosinophilic inflammatory infiltrates, with lesser numbers of mast cellsand neutrophils. The severity of these lesions was variable, however, insome areas, it progressed to intraepidermal eosinophilic pustulation andcollagen necrobiosis within the superficial dermis.

Taken together, the tissue samples indicated the presence of superficialperivascular/periadnexal, mastocytic and eosinophilic, subacutedermatitis. Lesions noted in all the slide specimens examined areconsistent with an allergic Type I hypersensitivity reaction.

Example 10

This example further demonstrates the ability of proteins described inExamples 2 and 3 to induce an allergic response in animals naturallysusceptible to flea allergy dermatitis through skin tests performed ondogs. These reactions were compared to those obtained using the currentstandard for diagnosis of flea allergy dermatitis, Greer Whole FleaExtract (Greer Laboratories, Inc., Lenoir, N.C.). In addition, in orderto determine specificity of the reactions, test results were compared tothose obtained from a population of control dogs with normal skin and apopulation of dogs with pruritic skin disorders other than flea allergydermatitis.

Three groups of dogs were used in the study: (1) 10 dogs with naturallyoccurring flea allergy dermatitis as determined by clinical signs,presence of fleas at the time of diagnosis, and a positive immediate ordelayed reaction to Greer Whole Flea Extract; (2) 10 dogs withnon-flea-related pruritic dermatoses including, but not limited to,atopy, food allergy dermatitis, pyoderma, seborrhea, and other parasitichypersensitivity reactions; and (3) 10 dogs with normal skin and nohistory of chronic skin diseases. The dogs were of any breed, age orsex. They were recruited from the hospital population of the Ohio StateUniversity Veterinary Teaching Hospital, Columbus, Ohio. All dogs hadwritten owner consent to participate in the study.

All tests and subjective scoring were performed by Kenneth W. Kwochka,D.V.M., Diplomat ACVD, (American College of Veterinary Dermatologists),in the Dermatology Examination Room at the Veterinary Teaching Hospital,College of Veterinary Medicine, The Ohio State University, Columbus,Ohio. All dogs were tested on the anterior-ventral-lateral aspect of thechest on the left side. Dogs were sedated for testing using standarddosages of xylazine and atropine administered intravenously immediatelybefore the skin test. No glucocorticoids, antihistamines, or othernon-steroidal antiinflamatory medications were allowed for at least 3weeks prior to testing. The area for testing was gently clipped with a#40 electric clipper blade and the injection sites marked with anindelible black felt-tipped marking pen. Twenty-two sites were marked:two rows of ten dots and one row of two dots. Intradermal injectionswere placed both above and below each mark for a total of forty-fourinjections that were administered in the following order:

Row 1: Neg. cont.-Histamine-Greer-Greer-Flea saliva-Flea saliva-A-A-B-B

Row 2: C1-C1-C2-C2-D1-D1-D2-D2-E-E

Row 3: F-F-G-G-H-H-I-I-J-J-

Row 4: K-K-L-L-M1-M1-M2-M2-N-N

Row 5: FS2-FS2

Row 6: Neg. cont.-Histamine

Each site was injected intradermally with 50 μl of sterile diluent (Neg.cont.), 1/100,000 w/v histamine phosphate (Histamine), Greer Whole FleaExtract (Greer), whole flea saliva (Flea saliva), or individual salivaryprotein fractions (fspA, (A); fspB, (B); fspC1, (C1); fspC2, (C2);fspD1, (D1); fspD2, (D2); fspE, (E); fspF, (F); fspG, (G); fspH, (H);fspI, (I); fspJ, (J); fspK, (K); fspL, (L); fspM1, (M1); fspM2, (M2);fspN, (N); and FS-2 (FS2). All injections were diluted in the samesterile diluent as the Neg. cont.

Skin reactions were read subjectively and objectively at 15 minutes and24 hours after injections. Owners were required to return their dogs tothe Veterinary Teaching Hospital for the 24 hour readings. Subjectiveassessments were basted on a scale of 0, 1+, 2+, 3+ and 4+ based onwheal size, amount of erythema and amount of induration. Objectiveassessment was based on wheal diameter measured in millimeters.

Comparison of Skin Reactions:

A. FAD Dogs:

Of the 10 dogs positive to Greer, 7 (70%) were positive the Flea Saliva(FS). None of the 3 FS-negative dogs reacted to any of the salivaryprotein fractions. Additionally, the 3 dogs negative to FS at 15 minuteswere negative to everything at 24 hours. The 7 FS-positive dogs wereused to summarize the 15 minute reactions, shown below in Table 11.

TABLE 11 Immediate (15 min) subjective scores of 7 FS-positive dogs totest antigens % Positive Scores ≧2+ Scores ≧3+ 0 I 14 B, I, J, L B, D1,J, L 29 A, C1, C2, D1 A, C1, C2, K 43 E, F, K D2, E, F, H, M2 57 D2, H,M2 G, N, FS2 71 G, M1 M1 86 N, FS2 Greer 100 Greer, FS FS

Four of the 7 FS-positive dogs could not be evaluated at 24 hoursbecause the severity of the immediate reactions warrantedantiinflammatory therapy. The remaining 3 FS-positive dogs were used tosummarize the 24 hour reactions, shown below in Table 12.

TABLE 12 Delayed (24 hr) subjective scores of 3 FS-positive dogs to testantigens % Positive Scores ≧2+ Scores ≧3+ 0 33 M2 67 Greer, FS, N, FS2FS, N, FS2 100

B. Normal Dogs:

Three dogs had an immediate reaction to the skin test antigens to someextent. None had a positive delayed reaction at 24 hours. A summary ofthe immediate (15 min) subjective results is shown below in Table 13.

TABLE 13 Immediate (15 min) subjective scores of 10 normal dogs to testantigens % Positive Scores ≧2+ Scores ≧3+ 0 10 N, FS2 FS2 20 Greer, FSGreer, FS 30 40 50 60 70 80 90 100Individual Dog Comments:Dog #1: Greer 3+, FS 3+, N 2+, FS2 4+Dog #2: Greer 3+Dog #3: FS 3+

C. Non-FAD Pruritis Dogs:

Six dogs had an immediate reaction to the skin test antigens to someextent. A summary of the immediate (15 min) subjective results is shownbelow in Table 14.

TABLE 14 Immediate (15 min) subjective scores of 10 Non-FAD pruritisdogs to test antigens % Positive Scores ≧2+ Scores ≧3+ 0 10 G, O G, O 20Greer, M1 Greer, FS, M1, M2 30 FS, M2, N N 40 50 60 70 80 90 100Individual Dog Comments:

Dog #1: FS 2+, M1 3+, M2 3+, N 3+, FS2 3+

-   -   Atopic dog under chronic flea exposure

Dog #2: FS 4+, G 4+, M1 4+, M2 3+, N 3+

-   -   Atopic dog under chronic flea exposure

Dog #3: FS 4+, M2 2+

-   -   Atopic dog under chronic flea exposure

Dog #4: N 3+

-   -   Atopic dog under chronic flea exposure

Dog #5: Greer 4+

-   -   Chronic otitis externa

Dog #6: Greer 4+

-   -   Generalized demodicosis (mange)    -   Dogs #1, #2 and #3 all came back to the clinic subsequently and        were diagnosed with FAD and were Greer positive.

Three dogs had a delayed reaction to the skin test antigens to someextent. A summary of the delayed (24 hr) subjective results is shownbelow in Table 15.

TABLE 15 Delayed (24 hr) subjective scores of 10 Non-FAD pruritis dogsto test antigens % Positive Scores ≧2+ Scores ≧3+ 0 10 FS, N, FS2 Greer,FS, N, FS2 20 30 Greer 40 50 60 70 80 90 100Individual Dog Comments:

Dog #3: Greer 2+

-   -   Atopic dog under chronic flea exposure

Dog #4: Greer 2+

-   -   Atopic dog under chronic flea exposure

Dog #6: Greer 3+, FS 3+, N 3+, FS2 3+

-   -   Generalized demodicosis (mange)

As an aid in determining the fraction(s) of flea saliva that correlatebest with a positive skin test result, all the data for the artificiallysensitized and clinically diagnosed FAD dogs that were 2+ or greater toFS (12 dogs total; 5 artificially sensitized and 7 clinically diagnosedas FAD positive) were tabulated according to the responses to the testantigens. The immediate (15 min) subjective results are shown below inTable 16, and the delayed (24 h) subjective results are shown below inTable 17.

TABLE 16 PERCENT RESPONDING (15 min subjective score) ArtificiallyClinical Sensitized (5) Diagnosis (7) Combined (12) Antigen Score ≧2+Score ≧3+ Score ≧2+ Score ≧3+ Score ≧2+ Score ≧3+ Greer 20 20 100 86 6758 FS 100 80 100 100 100 92 A 0 0 29 29 17 17 B 0 0 14 14 8 8 C 0 0 2929 17 17 D1 0 0 29 14 17 8 D2 0 0 57 43 33 25 E 40 20 43 43 42 33 F 4040 43 43 42 42 G 60 40 71 57 67 50 H 80 20 57 43 67 33 I 100 40 14 0 5017 J 100 40 14 14 50 25 K 80 20 43 29 58 25 L 20 20 14 14 17 17 M1 10060 71 71 83 67 M2 100 80 57 43 75 58 N 100 60 86 57 92 58 FS2 80 60 8657 83 58

TABLE 17 PERCENT RESPONDING (24 hr subjective score) ArtificiallySensitized Clinical (5) Diagnosis (7) Combined (12) Antigen Score ≧2+Score ≧3+ Score ≧2+ Score ≧3+ Score ≧2+ Score ≧3+ Greer 0 0 67 0 25 0 FS0 0 67 67 25 25 A 0 0 0 0 0 0 B 0 0 0 0 0 0 C 0 0 0 0 0 0 D1 0 0 0 0 0 0D2 0 0 0 0 0 0 E 0 0 0 0 0 0 F 0 0 0 0 0 0 G 0 0 0 0 0 0 H 0 0 0 0 0 0 I0 0 0 0 0 0 J 0 0 0 0 0 0 K 0 0 0 0 0 0 L 0 0 0 0 0 0 M1 20 0 0 0 13 0M2 0 0 33 0 13 0 N 20 0 67 67 38 25 FS2 60 20 67 67 63 38

The results of these studies indicate that the most substantialresponses were obtained for fractions fspG, fspH, fspM1, fspM2 and fspN.

Example 11

The following example illustrates the expression of fspI proteins inbacteria and in insect cells.

A. Expression of Flea Protein fspI in E. coli.

A 500 bp DNA fragment of fspI was PCR amplified from nucleic acidmolecule nfspI₅₉₁, using: Primer 11, a sense primer having the nucleicacid sequence 5′ ATTCGGATCCATGGAAAGTTAATAAAAAATGTAC 3′ (BamHI site inbold), denoted as SEQ ID NO:36; and Primer 12, an antisense primerhaving the nucleic acid sequence 5′ TAATGGATCCTTATTTTTTGGTCGACAATAAC 3′,denoted SEQ ID NO:37. The PCR product, a fragment of about 535nucleotides, denoted nfspI₅₃₅, was digested with BamHI restrictionendonuclease, gel purified, and subcloned into expression vectorpTrcHisB (available from InVitrogen Corp.) that had been digested withBamHI and CIP treated to produce recombinant molecule pHis-nfspI₅₃₅.

The recombinant molecule was transformed into both HB101 (available fromBRL, Gaithersburg, Md.) and BL21 (available from Novagen, Madison, Wis.)competent cells to form recombinant cells E.coliHB:pHis-nfspI₅₃₅ andE.coliBL:pHis-nfspI₅₃₅. The recombinant cells were cultured in anenriched bacterial growth medium containing 0.1 mg/ml ampicillin and0.1% glucose at 32° C. When the cells reached an OD₆₀₀ of about 0.4-0.5,expression was induced by the addition of 0.5 mM isopropylB-D-thiogalactoside (IPTG), and the cells were cultured for 2 hours at32° C.

SDS-polyacrylamide gel electrophoresis and western immunoblot analysesof recombinant cell lysates containing the fusion protein PHIS-PfspI₁₅₅were accomplished by standard procedures using either a T7 Tagmonoclonal antibody (available from InVitrogen Corp.) or rabbit anti-FADantiserum (#A3381) generated by Paravax, Inc. in Fort Collins, Colo.,produced by immunizing rabbits with nitrocellulose membranes containingflea saliva, produced as described in Example 2. Antigen/antibodyreactions were detected by colorimetric enzyme reactions using alkalinephosphatase-conjugated anti-mouse or anti-rabbit antibodies. A 28 kDprotein was detected on the immunoblots of induced lysates with bothprimary antibodies.

B. Expression of Flea Protein fspI in Insect Cells

Nucleic acid molecule nfspI₄₇₅ was PCR amplified from an fspI nucleicacid molecule using the following primers, which were designed tofacilitate expression in insect cells using a baculovirus vector: sensePrimer 13, with the BamHI site in bold, is 5′ CGC GGA TCC TAT AAA TATGGA GGA CAT CTG GAA AGT TAA TAA AAA ATG TAC ATC 3′, denoted as SEQ IDNO:44; and antisense Primer 14, with the XbaI site in bold, is 5′ GCTCTA GAG CAT TTA TTT TTT GGT CGA CAA TAA CAA AAC 3′, denoted as SEQ IDNO:45. The PCR product was digested with BamHI and XbaI and an about 475bp DNA fragment was excised and purified from an agarose gel.

Nucleic acid molecule nfspI₄₇₅ was ligated into a pVL1393 vector(available from InVitrogen Corp.) digested with BamHI and XbaI toproduce recombinant molecule pVL-nfspI₄₇₅.

The recombinant molecule was transfected into S. frugiperda Sf9 cellswith linearized Baculovirus DNA to form recombinant cell S.frugiperda:pVL-nfspI₄₇₅. The recombinant cells were cultured usingstandard conditions to produce recombinant virus. The transfectionsupernatant was also found to contain a 23 kD protein which reacted witha rabbit anti-FAD antiserum (#A3381) by Western blot analysis.

Example 12

This example describes the isolation of nucleic acid sequences encodingat least portions of flea saliva proteins in fspN, and theircharacterization relative to human prostatic acid phosphatase.

The flea salivary gland and the whole fed flea cDNA libraries describedpreviously in Example 6A were immunoscreened using New Zealand Whiterabbit antiserum developed against a collected mixture of flea salivaryproteins (e.g., the rabbit was immunized one or more times with a groundup nitrocellulose filter used as collection membrane to collect fleasaliva proteins, followed by one or more immunization with a flea salivaprotein extract eluted from a Duropore filter). The immunoscreeningprotocols used are those described in the picoBlue T Immunoscreening Kitinstruction manual, available from Stratagene, Inc. The methods forpreparation of the cDNA expression libraries for immunoscreening, i.e.,expression of the cDNA clones and procedures for transferring lambdaphage plaques to membranes for immunoscreening, are described in theZAP-cDNA Synthesis Kit instruction manual, also available fromStratagene, Inc., La Jolla, Calif.

Forty immunopositive clones were selected from the screening. Oneimmunopositive clone was derived from the salivary gland cDNA libraryand 39 other immunopositive clones were derived from the whole fed fleacDNA library. The initial fspN-protein cDNA sequences, termed nfspN(A)and nfspN(B) were isolated from the whole fed flea cDNA library and camefrom this initial immunoscreening.

Partial nucleotide sequences for nfspN(A) and nfspN(B) are representedby SEQ ID NO's. Each sequence represents approximately the carboxylterminal half of the cDNA gene coding region as well as the 3′untranslated region through the poly (A) region. The nucleotide sequencefor a nfspN(A) nucleic acid molecule named nfspN(A)₆₄₆ is denoted as SEQID NO:50. Translation of SEQ ID NO:50 yields a protein named PfspN(A)₁₇₂having an amino acid sequence denoted SEQ ID NO:51. The nucleotidesequences for a nfspN(B) nucleic acid molecule named nfspN(B)₆₁₂ isdenoted SEQ ID NO:52. Translation of SEQ ID NO:52 yields a protein namedPfspN(B)₁₅₃ having an amino acid sequence denoted SEQ ID NO:53.

In addition, an apparent N-terminal amino acid sequence deduced fromnucleic acid sequence of nfspN(A), named PfspN(A)₅₆ and denoted SEQ IDNO:54, was determined. The amino acid sequence of PfspN(A)₅₆ (i.e., SEQID NO:54) is similar, but not identical, to the N-terminal amino acidsequences obtained for fspN1 (SEQ ID NO:11), fspN2 (SEQ ID NO:12) andfspN3 (SEQ ID NO:13). While not being bound by theory, it is believedthat there is a family of fspN proteins that are found in flea saliva,which may be due to allelic variation or multiple genes in the fleagenome. Nucleic acid molecules nfspN(A)₆₄₆ and nfspN(B)₆₁₂ are about 76%identical, and the translated products are about 65% identical.

In a second immunoscreening experiment in which antiserum collected froma rabbit that was immunized with the proteins in peak N of the HPLCseparation of flea saliva extract described in Example 4 (i.e., fspNproteins) was used to probe a flea salivary gland cDNA library (preparedas described in Example 6), approximately 20 positive clones wereisolated. The nucleic acid sequence of one of the recovered nucleic acidmolecules appears to be identical to that of nfspN(A). At least two ofthe other nucleic acid molecules have nucleic acid sequences that aresimilar, but not identical, to that of nfspN(A), again supporting thelikelihood of a family of fspN proteins in flea saliva. Yet anothernucleic acid molecule appears to have a nucleic acid sequence that issimilar to myosin gene sequences.

The nucleic acid and amino acid sequences of the fspN(A) and fspN(B)nucleic acid molecules and proteins, respectively, were compared toknown nucleic acid and amino acid sequences using a Genbank homologysearch. Both nucleic acid sequences were found to be similar to thecorresponding (i.e., carboxyl-terminal) region of the nucleic acidsequence of human prostatic acid phosphatase. The most highly conservedregion of continuous similarity between flea and human amino acidsequences spans from about amino acid 272 through about amino acid 333of the human enzyme. Comparison of the nucleic acid sequence encodingamino acids from about 268 through about 333 of the human enzyme withthe corresponding regions of nfspN(A) and nfspN(B) nucleic acidsequences indicate that nfspN(A) is about 40% identical, and nfspN(B) isabout 43% identical, to that region of the human prostatic acidphosphatase gene. Comparison of the region spanning from about aminoacid 268 through about amino acid 333 of the human enzyme with thecorresponding regions of PfspN(A) and PfspN(B) indicate that PfspN(A) isabout 28% identical, and PfspN(B) is about 30% identical, to that regionof the human prostatic acid phosphatase gene. The possibility that atleast some fspN proteins encode an active acid phosphatase is supportedby the finding that flea saliva extract FS-3 has been shown to have acidphosphatase activity, as described in Example 3.

The apparent complete nucleic acid sequence of the coding region ofnucleic acid molecule nfspN(A), referred to herein as nfspN(A)₁₁₉₇, isdenoted herein as SEQ ID NO:55. Translation of SEQ ID NO:55 yields anapparent full-length fspN protein named PfspN(A)₃₉₈ having an amino acidsequence denoted herein as SEQ ID NO:56. (It should be noted thatalthough nucleic acid sequence SEQ ID NO:55 and amino acid sequence SEQID NO:56 do not exactly match nucleic acid sequence SEQ ID NO:50 oramino acid sequences SEQ ID NO:51 or SEQ ID NO:54 in the correspondingregions, the mismatches are likely due to sequencing errors in SEQ IDNO:50, SEQ ID NO:51 and SEQ ID NO:54.)

Comparison of SEQ ID NO:56 with the N-terminal amino acid sequencesobtained for fspN1 (SEQ ID NO:11), fspN2 (SEQ ID NO:12) and fspN3 (SEQID NO:13) indicates that the amino terminal amino acids of fspN1 andfspN2 correspond to amino acid position 18 of SEQ ID NO:56, while theamino terminal amino acid of fspN3 corresponds to amino acid position 20of SEQ ID NO:56. SEQ ID NO:13 appears to be identical to the region ofSEQ ID NO:56 spanning amino acid positions from 20 through 39,suggesting that nfspN(A) encodes fspN3. SEQ ID NO:11 is about 67%identical to the corresponding region of SEQ ID NO:56, and SEQ ID NO:12is about 60% identical (discounting the 3 unknown amino acids in SEQ IDNO:12) to the corresponding region of SEQ ID NO:56, supporting thesuggestion that fspN1 and fspN2 are members of the same flea salivaprotein family as fspN3.

Comparison of SEQ ID NO:56 with the amino acid sequence of humanprostatic acid phosphatase indicates that the two sequences share about30% identity at the amino acid level.

Example 13

This Example demonstrates the production of a bacterial recombinant cellincluding an fspN protein and use of that cell to produce the fspNprotein.

An about 1000 bp DNA fragment, denoted nfspN₁₀₀₀, was PCR amplified froma nucleic acid molecule encoding an fspN protein using the followingprimers: F7 sense, having nucleic acid sequence 5′GGCGTCTCGAGAGAATTGAAATTTGTGTTTGCG 3′ (XhoI site in bold), denoted SEQ IDNO:46; and F7 antisense, having nucleic acid sequence 5′AGACGAGAATTCCAATTTATCATGAGCGG 3′ (EcoRI site in bold), denoted SEQ IDNO:47. The PCR product was digested with XhoI and EcoRI restrictionendonucleases, gel purified and subcloned into expression vectorpTrcHisB (available from InVitrogen, Corp.) that had been digested withXhoI and EcoRI to form recombinant molecule pHis-nfspN₁₀₀₀. Therecombinant molecule was transformed into E. coli BL21 competent cells(available from Novagen) to form recombinant cell E.coli:pHis-nfspN₁₀₀₀.

Recombinant cell E. coli:pHis-nfspN₁₀₀₀ was cultured and induced asdescribed in Example 11A to produce fusion protein PHIS-PfspN3. Therecombinant fusion protein was detected by immunoblot analysis using theT7 Tag monoclonal antibody as described in Example 11A. PHIS-PfspN3 wasNi purified using Ni-NTA spin kit (available from Qiagen, Chatsworth,Calif.) and the purification verified using T7 Tag monoclonal antibodyas described above.

Example 14

The Example demonstrates the expression of an fspN protein in insectcells.

Recombinant molecule pVL-nfspN₁₀₀₀ containing the nfspN₁₀₀₀ nucleic acidmolecule operatively linked to baculovirus polyhedrin transcriptioncontrol sequences was produced in the following manner. An about 1000 bpDNA fragment, denoted nfspN₁₀₀₀, was PCR amplified from a nucleic acidmolecule encoding an fspN protein using the following primers: senseprimer 17, having nucleic acid sequence 5′ CCG GAA TTC CGG TAT AAA TATGTG GCG TCT ACT G 3′ (EcoRI site in bold), denoted SEQ ID NO:48, anddesigned to enhance expression in insect cells; and antisense primer 18,having nucleic acid sequence 5′ CCG GAA TTC TTA AGA CGA TTT ACA CAA TTTATC 3′ (EcoRI site in bold), denoted SEQ ID NO:49. The PCR product wasdigested with EcoRI and non-directionally cloned into the baculovirusshuttle vector pVL1393 (available from InVitrogen, Corp.). Orientationwas determined by restriction digest with the enzyme EcoRV. Theresultant recombinant molecule, i.e., pVL-nfspN₁₀₀₀, was co-transfectedinto S. frugiperda cells (donated by the Colorado Bioprocessing Center,Fort Collins, Colo.) with wild type linear baculovirus DNA (AcMNPV) andinsectin cationic liposome according to manufacturer's specifications(available from InVitrogen Corp.) to produce recombinant cell S.frugiperda:pVL-nfspN₁₀₀₀. The supernatant was tested five dayspost-transfection by Western blot analysis using rabbit antiserumagainst flea fspN proteins (as described in Example 12; denoted B2237)and a protein approximately 40 kD was detected. The recombinant virus,vBV-nfspN₁₀₀₀ was recovered from the supernatant and plaque purified.

Example 15

This Example demonstrates that use of ELISAs to detect anti-flea salivaIgE antibodies in the sera of dogs sensitized to fleas or flea saliva.

A. In a first study, sera collected from three dogs that had beenartificially sensitized to flea bites were pooled and pretreated bycontacting the pooled sera with Protein G to remove at least some of thenon-IgE immunoglobulins present in the sera. IgE antibodies were thenaffinity-purified from the pretreated sera using Con-A chromatography.

The affinity-purified IgE antibodies were exposed to the following fleasaliva products and proteins: FS-1 saliva extract at 2 mg/ml (23,300flea-hours per μl); fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspf,fspG, fspH, fspI, fspJ, fspK, fspL, fspM1, fspM2, and fspN (from a233,000 flea-hours per μl sample applied to HPLC chromatography asdescribed in Example 3). The flea saliva products and proteins weresuspended in 0.1 M sodium carbonate, pH 9.6, and 100 μl samples of eachwere placed in microtiter dish wells. The samples were incubatedovernight at room temperature, washed 5 times with PBS/Tween, blockedwith a solution of PBS, 2% BSA, 0.02% NaN₃, for 1 hour at 37° C., andwashed 5 times with PBS/Tween. The washed wells were each exposed to 100μl aliquots of the affinity-purified dog IgE antibodies for 1 hour at37° C. The wells were washed 5 times with PBS/Tween and exposed for 1hour, at 37° C., to 100 μl of a monoclonal mouse anti-canine IgEantibody preparation diluted 1:000 in PBS, 2% BSA, 0.05% Triton X-100.The wells were washed 5 times with PBS/Tween, exposed for 1 hour, at 37°C., to 100 μl donkey anti-mouse IgG (H+L)-HRP, and washed 5 times withPBS/Tween. The wells were developed with 100 μl KPL TMB:H₂O₂, 1:1, for10 minutes, the reaction being stopped with 50 μl 2.5 N hydrogensulfate. The wells were read at 450 nm.

The results, shown in Table 18 and FIG. 10, indicate that FAD+ dogs havein their sera IgE antibodies that react in a sensitive and specificmanner with FS-1 flea saliva extract as well as with flea salivaproteins fspE, fspF, fspG, fspH, fspI, fspJ, fspK, fspL, fspM1, fspM2and fspN. The IgE antibody preparation reacted minimally, if at all,with flea saliva proteins fspA, fspB, fspC1, fspC2, fspD1 and fspD2.Thus, the IgE reactivity closely followed the skin test results ofExample 8 in the artificially sensitized dogs with the same flea salivaproducts and proteins.

TABLE 18 Volume of Antigen Fraction 0.5 μl 0.25 μl 0.125 μl 0.063 μl A0.007 0.008 0.012 0.018 B 0.016 0.010 0.013 0.052 C1 0.035 0.008 0.0350.020 C2 0.022 0.009 0.002 0.005 D1 0.013 0.025 0.004 0.005 D2 0.0590.018 0.017 0.012 E 0.214 0.263 0.206 0.092 F 0.276 0.393 0.217 0.114 G0.288 0.217 −0.010 −0.010 H 0.503 0.336 0.203 0.062 I 1.076 0.997 0.9170.637 J 0.955 0.816 0.673 0.456 K 1.095 0.898 0.815 0.690 L 0.991 0.7210.485 0.162 M1 1.251 1.190 0.840 0.454 M2 1.561 1.105 0.902 0.558 N1.989 1.887 1.819 1.435 FS-1 1.367 1.246 0.982 0.604 none 0.002 0.0050.008 0.121B. In a second study, serum collected from a dog that had beenartificially sensitized to flea bites was pretreated by contacting theserum with Protein G to remove at least some of the non-IgEimmunoglobulins present in the serum. The reactivity of the pretreatedserum to FS-1 flea saliva extract was determined as described in Example15A. Also tested was the reactivity to FS-1 flea saliva extract of seracollected from dogs infected with heartworm, pooled and pretreated bycontacting the serum with Protein G. The results, shown in Table 19 andFIGS. 11A and 11B, demonstrate a dose dependent reactivity of IgE fromthe FAD+ dog while IgE from heartworm infected dogs had no reactivityagainst FS-1 flea saliva extract.

TABLE 19 Sera dil. 1:2 1:4 1:8 1:16 1:32 1:64 1:128 none DOG 2082128 2μg 1.67 1.20 0.85 0.57 0.34 0.19 0.11 0.01 1 μg 1.43 1.16 0.80 0.49 0.300.17 0.10 0.00 0.5 μg 1.32 1.02 0.71 0.46 0.28 0.14 0.08 0.00 0.25 μg1.18 0.92 0.59 0.38 0.22 0.12 0.06 0.00 0.13 μg 0.95 0.80 0.52 0.30 0.190.11 0.06 0.00 none 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.01 HEARTWORMPOOL 2 μg 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 μg 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.5 μg 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.25 μg 0.00 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.13 μg 0.01 0.00 0.000.00 0.00 0.00 0.00 0.00 none 0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00

Example 16

This example demonstrates the ability of formulations of the presentinvention, including formulations comprising flea saliva extract FS-1,E. coli-produced fspH, E. coli-produced fspN3 or insect cell S.frugiperda-produced fspN3 to identify animals susceptible to fleaallergy dermatitis (i.e., to induce flea allergy dermatitis in an animalsusceptible to flea allergy dermatitis).

The formulations were produced as follows. FS-1 was produced asdescribed in Example 2. E. coli-produced fspH was produced by an E. colicell transformed with a nucleic acid molecule encoding fspH operativelylinked to expression vector λP_(R)/T²ori/S10HIS-RSET-A9, the productionof which is described in PCT/US95/02941, Example 7; the resultantPHIS-fspH fusion protein was purified as described in Example 13. E.coli-produced fspN3 and S. frugiperda-produced fspN3 were produced asdescribed in Examples 13 and 14, respectively; E. coli-produced fspN3was purified as described in Example 11A; S. frugiperda-produced fspN3was purified by anion/cation exchange chromatography.

The formulations were tested in artificially sensitized dogs 2080109,2082101, 2082128, BFQ2, CPO2, CQQ2, as described in Example 8. Theinjected samples were as follows: (a) saline negative control; (b)histamine positive control; (c) 2 μg FS-1; (d) 0.1 μg E. coli-producedfspH; (e) 1.0 μg E. coli-produced fspH; (f) 0.2 μg E. coli-producedfspN3; (g) 2.0 μg E. coli-produced fspN3; (h) 0.2 μg S.frugiperda-produced fspN3; and (i) 2.0 μg S. frugiperda-produced fspN3.The immediate hypersensitivity results are shown in Table 20, and thedelayed hypersensitivity results are shown in Table 21. Scoring was asdescribed in Example 8; NA indicates a bad injection.

TABLE 20 a) Immediate (15 min.) subjective score (1-4+) (NA was a badinjection) E. coli E. coli S. frugiperda Sa- Hista- FS-H fspN3 fspN3 Dogline mine FS-1 0.1 1.0 0.2 2.0 0.2 2.0 109 0 4 4 NA 4 1 1 1 1 101 0 4 44 4 1 2 0 1 128 0 4 4 2 4 1 2 1 3 BFQ2 0 4 3 0 3 0 0 0 2 CPO2 0 4 4 4 41 4 1 2 CQQ2 0 4 4 4 4 0 0 0 2

TABLE 21 b) Delayed (24 hr.) subjective score (1-4+) (NA was a badinjection) E. coli E. coli S. frugiperda Sa- Hista- FS-H fspN3 fspN3 Dogline mine FS-1 0.1 1.0 0.2 2.0 0.2 2.0 109 0 0 0 NA 0 0 0 0 0 101 0 0 30 2 2 3 2 3 128 0 0 3 0 2 2 2 2 3 BFQ2 0 0 1 0 0 0 0 0 0 CPO2 0 0 0 0 00 0 0 0 CQQ2 0 0 0 0 0 0 0 0 0

In summary, these results indicate that E. coli-produced fspH exhibiteda strong positive immediate reaction in all dogs, the reaction beingproportional to the dogs' reaction to flea saliva. E. coli-produced andS. frugiperda-produced fspN3 proteins also exhibited a positiveimmediate reaction in 4 and all dogs, respectively. The two dogs thatshowed a strong delayed hypersensitive response reaction to FS-1 showedsimilar delayed hypersensitive response reactions to recombinantlyproduced fspH and fspN3.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. An isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of: (a) a nucleic acid sequence thatencodes a protein comprising SEQ ID NO:6; and (b) a nucleic acidsequence fully complementary to the nucleic acid sequence of (a).
 2. Theisolated nucleic acid molecule of claim 1, wherein said nucleic acidsequence is selected from the group consisting of: (a) a nucleic acidsequence that encodes a protein comprising SEQ ID NO:6, wherein theamino acid at position 10 of SEQ ID NO:6 is a cysteine, the amino acidat position 32 of SEQ ID NO:6 is a lysine, the amino acid at position 33of SEQ ID NO:6 is isoleucine, the amino acid at position 36 of SEQ IDNO:6 is isoleucine and the amino acid at position 37 of SEQ ID NO:6 iscysteine; and (b) a nucleic acid sequence fully complementary to thenucleic acid sequence of(a).
 3. An isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consistingof: (a) a nucleic acid sequence that encodes SEQ ID NO:25 or SEQ IDNO:26; and (b) a nucleic acid sequence fully complementary to thenucleic acid sequence of (a).